Hongmin Li

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• Li, Z., Zhang, J., & Li, H. (2017). Flavivirus NS2B/NS3 Protease: Structure, Function, and Inhibition. In Viral Proteases and Their Inhibitors. Academic Press. doi:10.1016/B978-0-12-809712-0.00007-1
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  Abstract Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis, hemorrhagic fever, and microcephaly, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to Flavivirus replication, such as the viral protease. During viral replication, the Flavivirus genome is translated as a single-polyprotein precursor, which must be cleaved into individual proteins by host proteases and a complex of the viral protease, NS3, and its cofactor, NS2B. As this cleavage is an obligate step of the viral life cycle, the Flavivirus protease is an attractive target for antiviral drug development. In this chapter, we will survey the flaviviral protease and recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from Flavivirus protease crystal structures.
• Li, H., Jin, L., Zeng, Z., Wang, M., & Wang, D. (1995). Crystal structure of an acidic neurotoxin from East Asia scorpion Buthus martensii Karsch. In Peptide: Biology & Chem.. Springer Netherlands. doi:10.1007/978-94-010-9069-8_28

Journals/Publications

• Samrat, S. K., Bashir, Q., Huang, Y., Trieshmann, C. W., Tharappel, A. M., Zhang, R., Chen, K., Zheng, Y. G., Li, Z., & Li, H. (2023).

  Broad-Spectrum Small-Molecule Inhibitors Targeting the SAM-Binding Site of Flavivirus NS5 Methyltransferase

  . ACS Infectious Diseases, 9(7), 1319-1333. doi:10.1021/acsinfecdis.2c00571
• Samrat, S. K., Bashir, Q., Zhang, R., Huang, Y., Liu, Y., Wu, X., Brown, T., Wang, W., Zheng, Y. G., Zhang, Q., Chen, Y., Li, Z., & Li, H. (2023).

  A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases

  . Emerging Microbes & Infections, 12(1). doi:10.1080/22221751.2023.2204164
• Li, H., Tharappel, A. M., Li, Z., Zhu, Y. C., Wu, X., Chaturvedi, S., & Zhang, Q. (2022). Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing. ACS Infectious Diseases, 8(9), 1851-1868. doi:10.1021/acsinfecdis.2c00137
• Li, Z., Li, H., Lang, Y., & Chen, K. (2021). The nucleocapsid protein of zoonotic betacoronaviruses is an attractive target for antiviral drug discovery.. Life sciences, 282, 118754. doi:10.1016/j.lfs.2020.118754
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  Betacoronaviruses are in one genera of coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), etc. These viruses threaten public health and cause dramatic economic losses. The nucleocapsid (N) protein is a structural protein of betacoronaviruses with multiple functions such as forming viral capsids with viral RNA, interacting with viral membrane protein to form the virus core with RNA, binding to several cellular kinases for signal transductions, etc. In this review, we highlighted the potential of the N protein as a suitable antiviral target from different perspectives, including structure, functions, and antiviral strategies for combatting betacoronaviruses.
• Zhao, J., Williams, J. E., Wang, C., Mills, K. V., Li, Z., Li, H., & Jaramillo, M. V. (2021). An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein.. Scientific reports, 11(1), 11680. doi:10.1038/s41598-021-91090-w
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  Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.
• Zhou, R., Zhou, J., Xue, Y., Xu, J., Shi, P. Y., & Li, H. (2021). Drug repurposing approach to combating coronavirus: Potential drugs and drug targets.. Medicinal research reviews, 41(3), 1375-1426. doi:10.1002/med.21763
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  In the past two decades, three highly pathogenic human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus, and, recently, SARS-CoV-2, have caused pandemics of severe acute respiratory diseases with alarming morbidity and mortality. Due to the lack of specific anti-CoV therapies, the ongoing pandemic of coronavirus disease 2019 (COVID-19) poses a great challenge to clinical management and highlights an urgent need for effective interventions. Drug repurposing is a rapid and feasible strategy to identify effective drugs for combating this deadly infection. In this review, we summarize the therapeutic CoV targets, focus on the existing small molecule drugs that have the potential to be repurposed for existing and emerging CoV infections of the future, and discuss the clinical progress of developing small molecule drugs for COVID-19.
• Bashir, Q., Li, Z., Li, H., Lemaster, D. M., & Hernandez, G. (2020). Crystal structure and transient dimerization for the FKBP12 protein from the pathogenic fungus Candida auris.. Biochemical and biophysical research communications, 525(4), 1103-1108. doi:10.1016/j.bbrc.2020.03.059
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  International concern over the recent emergence of Candida auris infections reflects not only its comparative ease of transmission and substantial mortality but the increasing level of resistance observed to all three major classes of antifungal drugs. Diminution in virulence has been reported for a wide range of fungal pathogens when the FK506-binding protein FKBP12 binds to that immunosuppressant drug and the binary complex then inhibits the fungal calcineurin signaling pathway. Structure-based drug design efforts have described modifications of FK506 which modestly reduce virulence for a number of fungal pathogens while also lessening the side effect of suppressing the tissue immunity response in the patient. To aid in such studies, we report the crystal structure of Candida auris FKBP12. As physiological relevance has been proposed for transient homodimerization interactions of distantly related fungal FKBP12 proteins, we report the solution NMR characterization of the homodimerization interactions of the FKBP12 proteins from both Candida auris and Candida glabrata.
• Tharappel, A. M., Samrat, S. K., Li, Z., & Li, H. (2020). Prospect of SARS-CoV-2 spike protein: Potential role in vaccine and therapeutic development.. Virus research, 288, 198141. doi:10.1016/j.virusres.2020.198141
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  The recent outbreak of the betacoronavirus SARS-CoV-2 has become a significant concern to public health care worldwide. As of August 19, 2020, more than 22,140,472 people are infected, and over 781,135 people have died due to this deadly virus. In the USA alone, over 5,482,602 people are currently infected, and more than 171,823 people have died. SARS-CoV-2 has shown a higher infectivity rate and a more extended incubation period as compared to previous coronaviruses. SARS-CoV-2 binds much more strongly than SARS-CoV to the same host receptor, angiotensin-converting enzyme 2 (ACE2). Previously, several methods to develop a vaccine against SARS-CoV or MERS-CoV have been tried with limited success. Since SARS-CoV-2 uses the spike (S) protein for entry to the host cell, it is one of the most preferred targets for making vaccines or therapeutics against SARS-CoV-2. In this review, we have summarised the characteristics of the S protein, as well as the different approaches being used for the development of vaccines and/or therapeutics based on the S protein.
• Tharappel, A. M., Samrat, S. K., Li, Z., & Li, H. (2020). Targeting Crucial Host Factors of SARS-CoV-2.. ACS infectious diseases, 6(11), 2844-2865. doi:10.1021/acsinfecdis.0c00456
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  Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide since its first incidence in Wuhan, China, in December 2019. Although the case fatality rate of COVID-19 appears to be lower than that of SARS and Middle East respiratory syndrome (MERS), the higher transmissibility of SARS-CoV-2 has caused the total fatality to surpass other viral diseases, reaching more than 1 million globally as of October 6, 2020. The rate at which the disease is spreading calls for a therapy that is useful for treating a large population. Multiple intersecting viral and host factor targets involved in the life cycle of the virus are being explored. Because of the frequent mutations, many coronaviruses gain zoonotic potential, which is dependent on the presence of cell receptors and proteases, and therefore the targeting of the viral proteins has some drawbacks, as strain-specific drug resistance can occur. Moreover, the limited number of proteins in a virus makes the number of available targets small. Although SARS-CoV and SARS-CoV-2 share common mechanisms of entry and replication, there are substantial differences in viral proteins such as the spike (S) protein. In contrast, targeting cellular factors may result in a broader range of therapies, reducing the chances of developing drug resistance. In this Review, we discuss the role of primary host factors such as the cell receptor angiotensin-converting enzyme 2 (ACE2), cellular proteases of S protein priming, post-translational modifiers, kinases, inflammatory cells, and their pharmacological intervention in the infection of SARS-CoV-2 and related viruses.
• Woods, D., Vangaveti, S., Sweeney, A. M., Stanger, M. J., Li, Z., Li, H., Lesassier, D. S., Lennon, C. W., Hardison, G. E., Egbanum, I., Belfort, M., & Bacot-davis, V. R. (2020). Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.. mBio, 11(4). doi:10.1128/mbio.01403-20
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  Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.
• Xu, J., Li, H., Zhou, J., & Shi, P. Y. (2020). Broad Spectrum Antiviral Agent Niclosamide and Its Therapeutic Potential.. ACS infectious diseases, 6(5), 909-915. doi:10.1021/acsinfecdis.0c00052
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  The recent outbreak of coronavirus disease 2019 (COVID-19) highlights an urgent need for therapeutics. Through a series of drug repurposing screening campaigns, niclosamide, an FDA-approved anthelminthic drug, was found to be effective against various viral infections with nanomolar to micromolar potency such as SARS-CoV, MERS-CoV, ZIKV, HCV, and human adenovirus, indicating its potential as an antiviral agent. In this brief review, we summarize the broad antiviral activity of niclosamide and highlight its potential clinical use in the treatment of COVID-19.
• Zhang, J., Xia, M., Trudeau, N., Tharappel, A. M., Samrat, S. K., Sakamuru, S., Rugenstein, N., Li, Z., Li, H., Lang, Y., Kuo, L., Kramer, L. D., Koetzner, C. A., Huang, R., Hu, S., D'brant, L., & Butler, D. (2020). Methylene blue is a potent and broad-spectrum inhibitor against Zika virus in vitro and in vivo.. Emerging microbes & infections, 9(1), 2404-2416. doi:10.1080/22221751.2020.1838954
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  Many flaviviruses including the Dengue virus (DENV), Zika virus (ZIKV), West Nile virus, Yellow Fever virus, and Japanese encephalitis virus are significant human pathogens, unfortunately without any specific therapy. Here, we demonstrate that methylene blue, an FDA-approved drug, is a broad-spectrum and potent antiviral against Zika virus and Dengue virus both in vitro and in vivo. We found that methylene blue can considerably inhibit the interactions between viral protease NS3 and its NS2B co-factor, inhibit viral protease activity, inhibit viral growth, protect 3D mini-brain organoids from ZIKV infection, and reduce viremia in a mouse model. Mechanistic studies confirmed that methylene blue works in both entry and post entry steps, reduces virus production in replicon cells and inhibited production of processed NS3 protein. Overall, we have shown that methylene blue is a potent antiviral for management of flavivirus infections, particularly for Zika virus. As an FDA-approved drug, methylene blue is well-tolerated for human use. Therefore, methylene blue represents a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.
• Zhou, J., Zhang, Q. Y., Zhang, J., Xu, J., Trudeau, N., Tharappel, A. M., Samrat, S. K., Rugenstein, N., Li, Z., Li, H., Lang, Y., Kuo, L., Kramer, L. D., Koetzner, C. A., Hu, S., Fan, X., D'brant, L., Chen, H., & Butler, D. (2020). JMX0207, a Niclosamide Derivative with Improved Pharmacokinetics, Suppresses Zika Virus Infection Both In Vitro and In Vivo.. ACS infectious diseases, 6(10), 2616-2628. doi:10.1021/acsinfecdis.0c00217
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  Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207 showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B, better inhibition of viral protease function, and enhanced antiviral efficacy in the cell-based antiviral assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced viremia in a Zika virus (ZIKV) animal model. In summary, a niclosamide derivative, JMX0207, was identified, which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.
• Cho, J. J., Xu, Z., Parthasarathy, U., Drashansky, T. T., Helm, E. Y., Zuniga, A. N., Lorentsen, K. J., Mansouri, S., Cho, J. Y., Edelmann, M. J., Duong, D. M., Gehring, T., Seeholzer, T., Krappmann, D., Uddin, M. N., Califano, D., Wang, R. L., Jin, L., Li, H., , Lv, D., et al. (2019). Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.. Nature communications, 10(1), 701. doi:10.1038/s41467-019-08605-3
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  Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.
• Green, C. M., Li, Z., Smith, A. D., Novikova, O., Bacot-davis, V. R., Gao, F., Hu, S., Banavali, N. K., Thiele, D. J., Li, H., & Belfort, M. (2019). Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.. PLoS biology, 17(10), e3000104. doi:10.1371/journal.pbio.3000104
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  The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.
• Lang, Y., Li, Z., & Li, H. (2019). Analysis of Protein-Protein Interactions by Split Luciferase Complementation Assay.. Current protocols in toxicology, 82(1), e90. doi:10.1002/cptx.90
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  Protein-protein interactions are important in human disease. Developing and refining tools to understand physical contacts between signaling proteins is crucial. This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed. Step-by-step instructions are provided for performing these assays using the NS2B-NS3 interaction as an example. NS2B is an essential cofactor for flaviviral NS3 protease function. Advantages and disadvantages of these assays are further discussed. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Expression and purification of fusion proteins Basic Protocol 2: Analysis of prey/bait pairs by SLC-based NS2B-NS3 interaction assay Support Protocol 1: Interaction specificity assay Support Protocol 2: Competition binding assay: Dose-response inhibition using cold prey or bait Support Protocol 3: Competition binding assay: Inhibition by MBP-NS3 versus irrelevant MBP tag Support Protocol 4: SLC-based NS2B-NS3 interaction assay using NS2B mutations known to disrupt NS2B-NS3 interactions.
• Li, H., Ding, X., Zhang, Q. Y., Zhang, Q., Fan, X., & Ding, X. (2019). Contributions of Hepatic and Intestinal Metabolism to the Disposition of Niclosamide, a Repurposed Drug with Poor Bioavailability.. Drug metabolism and disposition: the biological fate of chemicals, 47(7), 756-763. doi:10.1124/dmd.119.086678
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  Niclosamide, an antiparasitic, has been repositioned as a potential therapeutic drug for systemic diseases based on its antiviral, anticancer, and anti-infection properties. However, low bioavailability limits its in vivo efficacy. Our aim was to determine whether metabolic disposition by microsomal P450 enzymes in liver and intestine influences niclosamide's bioavailability in vivo, by comparing niclosamide metabolism in wild-type, liver-Cpr-null (LCN), and intestinal epithelium-Cpr-null (IECN) mice. In vitro stability of niclosamide in microsomal incubations was greater in the intestine than in liver in the presence of NADPH, but it was much greater in liver than in intestine in the presence of UDPGA. NADPH-dependent niclosamide metabolism and hydroxy-niclosamide formation were inhibited in hepatic microsomes of LCN mice, but not IECN mice, compared with wild-type mice. In intestinal microsomal reactions, hydroxy-niclosamide formation was not detected, but rates of niclosamide-glucuronide formation were ∼10-fold greater than in liver, in wild-type, LCN, and IECN mice. Apparent Km and V max values for microsomal niclosamide-glucuronide formation showed large differences between the two tissues, with the intestine having higher Km (0.47 μM) and higher V max (15.8) than the liver (0.09 μM and 0.75, respectively). In vivo studies in LCN mice confirmed the essential role of hepatic P450 in hydroxy-niclosamide formation; however, pharmacokinetic profiles of oral niclosamide were only minimally changed in LCN mice, compared with wild-type mice, and the changes seem to reflect the compensatory increase in hepatic UDP-glucuronosyltransferase activity. SIGNIFICANCE STATEMENT: These results suggest that efforts to increase the bioavailability of niclosamide by blocking its metabolism by P450 enzymes will unlikely be fruitful. In contrast, inhibition of niclosamide glucuronidation in both liver and intestine may prove effective for increasing niclosamide's bioavailability, thereby making it practical to repurpose this drug for treating systemic diseases.
• Li, Z., Fu, B., Green, C. M., Liu, B., Zhang, J., Lang, Y., Chaturvedi, S., Belfort, M., Liao, G., & Li, H. (2019). Cisplatin protects mice from challenge of Cryptococcus neoformans by targeting the Prp8 intein.. Emerging microbes & infections, 8(1), 895-908. doi:10.1080/22221751.2019.1625727
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  The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essential and non-redundant cellular functions, a Prp8 intein inhibitor is a mechanistically novel antifungal agent. In this report, we demonstrated that cisplatin, an FDA-approved cancer drug, significantly arrested growth of Prp8 intein-containing fungi C. neoformans and C. gattii, but only poorly inhibited growth of intein-free Candida species. These results suggest that cisplatin arrests fungal growth through specific inhibition of the Prp8 intein. Cisplatin was also found to significantly inhibit growth of C. neoformans in a mouse model. Our results further showed that cisplatin inhibited Prp8 intein splicing in vitro in a dose-dependent manner by direct binding to the Prp8 intein. Crystal structures of the apo- and cisplatin-bound Prp8 inteins revealed that two degenerate cisplatin molecules bind at the intein active site. Mutation of the splicing-site residues led to loss of cisplatin binding, as well as impairment of intein splicing. Finally, we found that overexpression of the Prp8 intein in cryptococcal species conferred cisplatin resistance. Overall, these results indicate that the Prp8 intein is a novel antifungal target worth further investigation.
• Pearson, C. S., Nemati, R., Liu, B., Zhang, J., Scalabrin, M., Li, Z., Li, H., Fabris, D., Belfort, M., & Belfort, G. (2019). Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight.. Biotechnology and bioengineering, 116(4), 709-721. doi:10.1002/bit.26875
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  We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.
• Zhao, J., Ciulla, D. A., Xie, J., Wagner, A. G., Castillo, D. A., Zwarycz, A. S., Lin, Z., Beadle, S., Giner, J., Li, Z., Li, H., Callahan, B. P., Wang, C., & Banavali, N. K. (2019). General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.. Journal of the American Chemical Society, 141(46), 18380-18384. doi:10.1021/jacs.9b08914
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  Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 × 103 and 3.7 × 103 M-1 s-1, respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.
• Kelley, D. S., Lennon, C. W., Li, Z., Miller, M. R., Banavali, N. K., Li, H., & Belfort, M. (2018). Mycobacterial DnaB helicase intein as oxidative stress sensor.. Nature Communications, 9(1), 4363-4363. doi:10.2210/pdb6bs8/pdb
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  Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Here, we describe two inteins within one protein, the Mycobacterium smegmatis replicative helicase DnaB. These inteins, DnaBi1 and DnaBi2, have homology to inteins in pathogens, splice with vastly varied rates, and are differentially responsive to environmental stressors. Whereas DnaBi1 splicing is reversibly inhibited by oxidative and nitrosative insults, DnaBi2 is not. Using a reporter that measures splicing in a native intein-containing organism and western blotting, we show that H2O2 inhibits DnaBi1 splicing in M. smegmatis. Intriguingly, upon oxidation, the catalytic cysteine of DnaBi1 forms an intramolecular disulfide bond. We report a crystal structure of the class 3 DnaBi1 intein at 1.95 A, supporting our findings and providing insight into this splicing mechanism. We propose that this cysteine toggle allows DnaBi1 to sense stress, pausing replication to maintain genome integrity, and then allowing splicing immediately when permissive conditions return.
• Li, H., Zhang, Q. Y., Zhou, J., Zhang, Q., Zhang, J., Xia, M., Sakamuru, S., Qin, C., Li, Z., Kramer, L. D., Koetzner, C. A., Huang, R., Chen, H., & Brecher, M. (2018). Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease.. Antiviral research, 150, 217-225. doi:10.1016/j.antiviral.2017.12.018
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  Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC50 in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.
• Brecher, M., Li, Z., Liu, B., Zhang, J., Koetzner, C. A., Alifarag, A., Jones, S. A., Lin, Q., Kramer, L. D., & Li, H. (2017). A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.. PLoS pathogens, 13(5), e1006411. doi:10.1371/journal.ppat.1006411
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  The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.
• Li, Z., Brecher, M., Zhang, J., Sakamuru, S., Liu, B., Huang, R., Koetzner, C. A., Allen, C. A., Jones, S. A., Chen, H., Zhang, N., Tian, M., Gao, F., Lin, Q., Zhou, J., Xia, M., Kramer, L. D., Qin, C., Li, H., , Deng, Y. Q., et al. (2017). Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction.. Cell research, 27(8), 1046-1064. doi:10.1038/cr.2017.88
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  Recent outbreaks of Zika virus (ZIKV) highlight an urgent need for therapeutics. The protease complex NS2B-NS3 plays essential roles during flaviviral polyprotein processing, and thus represents an attractive drug target. Here, we developed a split luciferase complementation-based high-throughput screening assay to identify orthosteric inhibitors that directly target flavivirus NS2B-NS3 interactions. By screening a total of 2 816 approved and investigational drugs, we identified three potent candidates, temoporfin, niclosamide, and nitazoxanide, as flavivirus NS2B-NS3 interaction inhibitors with nanomolar potencies. Significantly, the most potent compound, temoporfin, not only inhibited ZIKV replication in human placental and neural progenitor cells, but also prevented ZIKV-induced viremia and mortality in mouse models. Structural docking suggests that temoporfin potentially binds NS3 pockets that hold critical NS2B residues, thus inhibiting flaviviral polyprotein processing in a non-competitive manner. As these drugs have already been approved for clinical use in other indications either in the USA or other countries, they represent promising and easily developed therapies for the management of infections by ZIKV and other flaviviruses.
• Pearson, C. S., Green, C. M., Li, Z., Zhang, J., Belfort, G., Shekhtman, A., Li, H., Belfort, M., & Chan, H. K. (2016). Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy.. The Journal of biological chemistry, 291(43), 22661-22670. doi:10.1074/jbc.m116.747824
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  Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH3)2Cl2], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.
• Brecher, M. B., Li, Z., Zhang, J., Chen, H., Lin, Q., Liu, B., & Li, H. (2015). Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase.. Protein science : a publication of the Protein Society, 24(1), 117-28. doi:10.1002/pro.2594
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  Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.
• Brecher, M., Chen, H., Li, Z., Banavali, N. K., Jones, S. A., Zhang, J., Kramer, L. D., & Li, H. (2015). Identification and Characterization of Novel Broad-Spectrum Inhibitors of the Flavivirus Methyltransferase.. ACS infectious diseases, 1(8), 340-9. doi:10.1021/acsinfecdis.5b00070
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  Flavivirus methyltransferase (MTase) is essential for viral replication. Here we report the identification of small molecules through virtual screening that putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function. Six of these computationally predicted binders were identified to show significant MTase inhibition with low micromolar inhibitory activity. The most active compounds showed broad-spectrum activity against the MTase proteins of other flaviviruses. Two of these compounds also showed low cytotoxicity and high antiviral efficacy in cell-based assays. Competitive binding analyses indicated that the inhibitors performed their inhibitory function through competitive binding to the SAM cofactor binding site of the MTase. The crystal structure of the MTase-inhibitor complex further supports the mode of action and provides routes for their further optimization as flavivirus MTase inhibitors.
• Brecher, M., Chen, H., Liu, B., Banavali, N. K., Jones, S. A., Zhang, J., Li, Z., Kramer, L. D., & Li, H. (2015). Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase.. PloS one, 10(6), e0130062. doi:10.1371/journal.pone.0130062
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  The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition.
• Califano, D., Cho, J. J., Uddin, M. N., Lorentsen, K. J., Yang, Q., Bhandoola, A., Li, H., & Avram, D. (2015). Transcription Factor Bcl11b Controls Identity and Function of Mature Type 2 Innate Lymphoid Cells.. Immunity, 43(2), 354-68. doi:10.1016/j.immuni.2015.07.005
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  Type 2 innate lymphoid cells (ILC2s) promote anti-helminth responses and contribute to allergies. Here, we report that Bcl11b, previously considered a T-cell-specific transcription factor, acted directly upstream of the key ILC2 transcription factor Gfi1 to maintain its expression in mature ILC2s. Consequently, Bcl11b(-/-) ILC2s downregulated Gata3 and downstream genes, including Il1rl1 (encoding IL-33 receptor), and upregulated Rorc and type 3 ILC (ILC3) genes. Additionally, independent of Gfi1, Bcl11b directly repressed expression of the gene encoding the ILC3 transcription factor Ahr, further contributing to silencing of ILC3 genes in ILC2s. Thus, Bcl11b(-/-) ILC2s lost their functions and gained ILC3 functions, and although they expanded in response to the protease allergen papain, they produced ILC3 but not ILC2 cytokines and caused increased airway infiltration of neutrophils instead of eosinophils. Our results demonstrate that Bcl11b is more than just a T-cell-only transcription factor and establish that Bcl11b sustains mature ILC2 genetic and functional programs and lineage fidelity.
• Lemaster, D. M., Mustafi, S. M., Brecher, M., Zhang, J., Heroux, A., Li, H., & Hernandez, G. (2015). Coupling of Conformational Transitions in the N-terminal Domain of the 51-kDa FK506-binding Protein (FKBP51) Near Its Site of Interaction with the Steroid Receptor Proteins.. The Journal of biological chemistry, 290(25), 15746-15757. doi:10.1074/jbc.m115.650655
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  Interchanging Leu-119 for Pro-119 at the tip of the β4-β5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β4-β5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β2 and β3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β2 and β3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.
• Xie, J., Xia, K., Singh, A. V., Tou, E., Li, L., Li, H., Wan, L. Q., Wang, C., Owen, T. S., Callahan, B. P., & Arduini, B. L. (2015). Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.. The Journal of biological chemistry, 290(18), 11591-600. doi:10.1074/jbc.m114.623264
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  Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a Ki of 2 μm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.
• Chen, H., Mustafi, S. M., Lemaster, D. M., Li, Z., Heroux, A., Li, H., & Hernandez, G. (2014). Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.. Acta crystallographica. Section D, Biological crystallography, 70(Pt 3), 636-46. doi:10.1107/s1399004713032112
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  The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2₁ and P3₁21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3₁21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P2₁ crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.
• Gamari, B. D., Zhang, D., Buckman, R. E., Milas, P., Denker, J. S., Chen, H., Li, H., & Goldner, L. S. (2014). Inexpensive electronics and software for photon statistics and correlation spectroscopy.. American journal of physics, 82(7), 708-722. doi:10.1119/1.4869188
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  Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors.
• Mustafi, S. M., Brecher, M., Zhang, J., Li, H., Lemaster, D. M., & Hernandez, G. (2014). Structural basis of conformational transitions in the active site and 80's loop in the FK506-binding protein FKBP12.. The Biochemical journal, 458(3), 525-36. doi:10.1042/bj20131429
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  The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80's loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80's loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans-cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80's loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80's loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50's loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue.
• Brecher, M., Zhang, J., & Li, H. (2013). The flavivirus protease as a target for drug discovery.. Virologica Sinica, 28(6), 326-36. doi:10.1007/s12250-013-3390-x
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  Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.
• Chen, H., Liu, L., Jones, S. A., Kass, J., Li, Z., Zhang, J., Kramer, L. D., Ghosh, A. K., Li, H., & Banavali, N. K. (2013). Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs.. Antiviral research, 97(3), 232-9. doi:10.1016/j.antiviral.2012.12.012
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  The flavivirus methyltransferase (MTase) sequentially methylates the N-7 and 2'-O positions of the viral RNA cap (GpppA-RNA→m(7)GpppA-RNA→m(7)GpppAm-RNA), using S-adenosyl-l-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC50 values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture.
• Chen, H., Zhou, B., Brecher, M., Jones, S. A., Li, Z., Zhang, J., Kramer, L. D., Ghosh, A. K., Li, H., Nag, D. K., & Banavali, N. K. (2013). S-adenosyl-homocysteine is a weakly bound inhibitor for a flaviviral methyltransferase.. PloS one, 8(10), e76900. doi:10.1371/journal.pone.0076900
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  The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.
• Li, H., Zhang, H., & Fine, C. H. (2013). Dynamic Business Share Allocation in a Supply Chain with Competing Suppliers. Operations Research, 61(2), 280-297. doi:10.1287/opre.1120.1155
• Li, Y., Chen, X., Wang, Z., Zhao, D., Chen, H., Chen, W., Zhou, Z., Zhang, J., Zhang, J., Li, H., & Chen, C. (2013). The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1.. Neoplasia (New York, N.Y.), 15(1), 39-48. doi:10.1593/neo.121362
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  Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) is an E3 ubiquitin ligase with unknown functions. Here, we show that HECTD3 confers cancer cell resistance to cisplatin. To understand the molecular mechanisms, we performed a yeast two-hybrid analysis and identified mucosa-associated lymphoid tissue 1 (MALT1) as an HECTD3-interacting protein. HECTD3 promotes MALT1 ubiquitination with nondegradative polyubiquitin chains by direct interacting with the MALT1 through its N-terminal destruction of cyclin domain. HECTD3 does not target MALT1 for degradation but stabilize it. HECTD3 depletion dramatically decreases the levels of MALT1 in MCF7 and HeLa cells treated with cisplatin, which is correlated to an increase in apoptosis. Knockdown of MALT1 likewise increases cisplatin-induced apoptosis in these cancer cells. However, HECTD3 over-expression leads to a decreased cisplatin-induced apoptosis, whereas overexpression of MALT1 partially rescues HECTD3 depletion-induced apoptosis. These findings suggest that HECTD3 promotes cell survival through stabilizing MALT1. Our data have important implications in cancer therapy by providing novel molecular targets.
• Li, Y., Kong, Y., Zhou, Z., Chen, H., Wang, Z., Hsieh, Y., Zhao, D., Zhi, X., J, H., Zhang, J., Li, H., Chen, C., Yc, H., X, Z., & Huang, J. (2013). The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8.. Cell death & disease, 4(11), e935. doi:10.1038/cddis.2013.464
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  Apoptosis resistance is a hurdle for cancer treatment. HECTD3, a new E3 ubiquitin ligase, interacts with caspase-8 death effector domains and ubiquitinates caspase-8 with K63-linked polyubiquitin chains that do not target caspase-8 for degradation but decrease the caspase-8 activation. HECTD3 depletion can sensitize cancer cells to extrinsic apoptotic stimuli. In addition, HECTD3 inhibits TNF-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 cleavage in an E3 ligase activity-dependent manner. Mutation of the caspase-8 ubiquitination site at K215 abolishes the HECTD3 protection from TRAIL-induced cleavage. Finally, HECTD3 is frequently overexpressed in breast carcinomas. These findings suggest that caspase-8 ubiquitination by HECTD3 confers cancer cell survival.
• Liu, L., Chen, H., Brecher, M. B., Li, Z., Wei, B., Nandi, B., Zhang, J., Ling, H., Braun, J., Li, H., & Winslow, G. M. (2013). Pfit is a structurally novel Crohn's disease-associated superantigen.. PLoS pathogens, 9(12), e1003837. doi:10.1371/journal.ppat.1003837
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  T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the pfiT gene of Pseudomonas fluorescens, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of pfiT at 1.7Å resolution and provide a functional analysis of the interaction of pfiT and its homolog, PA2885, with human class II MHC. Both pfiT and PA2885 bound to mammalian cells and stimulated the proliferation of human lymphocytes. This binding was greatly inhibited by anti-class II MHC HLA-DR antibodies, and to a lesser extent, by anti HLA-DQ and DP antibodies, indicating that the binding was class II MHC-specific. GST-pfiT efficiently precipitated both endogenous and in vitro purified recombinant HLA-DR1 molecules, indicating that pfiT directly interacted with HLA-DR1. Competition studies revealed that pfiT and the superantigen Mycoplasma arthritidis mitogen (MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that pfiT belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of pfiT indicates that it represents a new family of T cell superantigens.
• Mustafi, S. M., Chen, H., Li, H., Lemaster, D. M., & Hernandez, G. (2013). Analysing the visible conformational substates of the FK506-binding protein FKBP12.. The Biochemical journal, 453(3), 371-80. doi:10.1042/bj20130276
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  The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80's loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the β-sheet, across the active site to the α-helix and into the 50's loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80's loop. Line-broadening was also decreased for the residues in the α-helix and 50's loop, whereas line-broadening in the 40's loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40's loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.
• Alturaihi, H., Yacoub, D., Liu, L., Guo, W., Leveille, C., Jung, D., Khzam, L. B., Merhi, Y., Wilkins, J. A., Li, H., Mourad, W., & Fakhry, Y. E. (2012). Functional interaction of CD154 protein with α5β1 integrin is totally independent from its binding to αIIbβ3 integrin and CD40 molecules.. The Journal of biological chemistry, 287(22), 18055-66. doi:10.1074/jbc.m111.333989
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  In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.
• Dong, H., Liu, L., Zou, G., Zhao, Y., Li, Z., Lim, S. P., Li, H., & Shi, P. Y. (2010). Structural and functional analyses of a conserved hydrophobic pocket of flavivirus methyltransferase.. The Journal of biological chemistry, 285(42), 32586-95. doi:10.1074/jbc.m110.129197
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  The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2'-O positions of the viral RNA cap (GpppA-RNA → m(7)GpppA-RNA → m(7)GpppAm-RNA), using S-adenosyl-l-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-Å resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus, SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.
• Liu, L., Dong, H., Chen, H., Zhang, J., Ling, H., Li, Z., Li, H., & Shi, P. Y. (2010). Flavivirus RNA cap methyltransferase: structure, function, and inhibition.. Frontiers in biology, 5(4), 286-303. doi:10.1007/s11515-010-0660-y
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  Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5' terminal cap 1 structure (m(7)GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m(7)GpppA → m(7)GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.
• Liu, L., Li, Z., Guo, Y., Vanvranken, S. J., Mourad, W., & Li, H. (2010). Crystal structure of the Mycoplasma arthritidis-derived mitogen in apo form reveals a 3D domain-swapped dimer.. Journal of molecular biology, 399(3), 367-76. doi:10.1016/j.jmb.2010.04.030
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  Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vbeta elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-A resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the "reconstituted" MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM(wt) molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.
• Dong, H., Ren, S., Li, H., & Shi, P. Y. (2008). Separate molecules of West Nile virus methyltransferase can independently catalyze the N7 and 2'-O methylations of viral RNA cap.. Virology, 377(1), 1-6. doi:10.1016/j.virol.2008.04.026
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  West Nile virus methyltransferase catalyzes N7 and 2'-O methylations of the viral RNA cap (GpppA-RNA-->m(7)GpppAm-RNA). The two methylation events are independent, as evidenced by efficient N7 methylation of GpppA-RNA-->m(7)GpppA-RNA and GpppAm-RNA-->m(7)GpppAm-RNA, and by the 2'-O methylation of GpppA-RNA-->GpppAm-RNA and m(7)GpppA-RNA-->m(7)GpppAm-RNA. However, the 2'-O methylation activity prefers substrate m(7)GpppA-RNA to GpppA-RNA, thereby determining the dominant methylation pathway as GpppA-RNA-->m(7)GpppA-RNA-->m(7)GpppAm-RNA. Mutant enzymes with different methylation defects can trans complement one another in vitro. Furthermore, sequential treatment of GpppA-RNA with distinct methyltransferase mutants generates fully methylated m(7)GpppAm-RNA, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.
• Dong, H., Ren, S., Zhang, B., Zhou, Y., Puig-basagoiti, F., Li, H., & Shi, P. Y. (2008). West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism.. Journal of virology, 82(9), 4295-307. doi:10.1128/jvi.02202-07
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  Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA-->m(7)GpppA-RNA-->m(7)GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2'-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2'-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2'-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m(7)GpppA-RNA, after which the m(7)G moiety is repositioned to the GTP-binding pocket to register the 2'-OH of the adenosine with SAM, generating m(7)GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.
• Dong, H., Ray, D., Ren, S., Zhang, B., Puig-basagoiti, F., Takagi, Y., Ho, C. K., Li, H., & Shi, P. Y. (2007). Distinct RNA elements confer specificity to flavivirus RNA cap methylation events.. Journal of virology, 81(9), 4412-21. doi:10.1128/jvi.02455-06
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  The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
• Lemaster, D. M., Anderson, J. S., Wang, L., Guo, Y., Li, H., & Hernandez, G. (2007). NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin.. BMC structural biology, 7(1), 81. doi:10.1186/1472-6807-7-81
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  Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions..Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The differences in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 A and 1.04 A, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual molecules in the crystallographic asymmetric unit (approximately 0.3 A), indicating consistency with the "cut-and-paste" structuring of the hybrid rubredoxins in both crystal and solution..Each of the significant energetic interactions in the metal binding site-swapped hybrids appears to exhibit a 1-to-1 correspondence with the interactions present in the corresponding parental rubredoxin structure, thus providing a structural basis for the observed additivity in conformational stability and dynamics. The congruence of these X-ray and NMR experimental data offers additional support for the interpretation that the conventional treatment of NOE distance restraints contributes substantially to the systematic differences that are commonly reported between NMR- and X-ray-derived protein structures.
• Li, H., Zhao, Y., Guo, Y., Li, Z., Mourad, W., & Eisele, L. E. (2007). Zinc induces dimerization of the class II major histocompatibility complex molecule that leads to cooperative binding to a superantigen.. The Journal of biological chemistry, 282(9), 5991-6000. doi:10.1074/jbc.m608482200
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  Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vbeta elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn(2+) is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn(2+). However, in the presence of Zn(2+), a dimerized MAM/HLA-DR1/HA complex can arise through the Zn(2+)-induced DR1 dimer. In the presence of Zn(2+), cooperative binding of MAM to the DR1 dimer was also observed.
• Li, H., Zhao, Y., Guo, Y., Vanvranken, S. J., Li, Z., Mourad, W., & Eisele, L. E. (2007). Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen.. Molecular immunology, 44(5), 763-73. doi:10.1016/j.molimm.2006.04.010
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  Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn2+. A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn2+ to MAM solution resulted in a decrease of melting point (Tm), whereas addition of EDTA did not affect the Tm of MAM. These results imply that there is no defined Zn2+-binding site on MAM.
• Wang, L., Zhao, Y., Li, Z., Guo, Y., Jones, L. L., Kranz, D. M., Mourad, W., & Li, H. (2007). Crystal structure of a complete ternary complex of TCR, superantigen and peptide-MHC.. Nature structural & molecular biology, 14(2), 169-71. doi:10.1038/nsmb1193
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  'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.
• Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., Guo, Y., Bernard, K. A., Li, H., & Shi, P. Y. (2007). Structure and function of flavivirus NS5 methyltransferase.. Journal of virology, 81(8), 3891-903. doi:10.1128/jvi.02704-06
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  The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.
• Guo, Y., Li, Z., Vranken, S. J., & Li, H. (2006). A single point mutation changes the crystallization behavior of Mycoplasma arthritidis-derived mitogen.. Acta crystallographica. Section F, Structural biology and crystallization communications, 62(Pt 3), 238-41. doi:10.1107/s1744309106003691
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  Mycoplasma arthritidis-derived mitogen (MAM) functions as a conventional superantigen (SAg). Although recombinant MAM has been crystallized by the hanging-drop vapour-diffusion method, the crystals diffracted poorly to only 5.0 A resolution, with large unit-cell parameters a = 163.8, b = 93.0, c = 210.9 A, beta = 93.7 degrees in the monoclinic space group P2(1). Unit-cell content analysis revealed that as many as 24 molecules could be present in the asymmetric unit. Systematic alanine mutagenesis was applied in order to search for mutants that give crystals of better quality. Two mutants, L50A and K201A, were crystallized under the same conditions as wild-type MAM (MAMwt). Crystals of the L50A mutant are isomorphous with those of MAMwt, while a new crystal form was obtained for the K201 mutant, belonging to the cubic space group P4(1)32 with unit-cell parameters a = b = c = 181.9 A. Diffraction data were collected to 3.6 and 2.8 A resolution from crystals of the MAM L50A and K201A mutants, respectively. Molecular-replacement calculations suggest the presence of two molecules in the asymmetric unit for the MAM K201A mutant crystal, resulting in a VM of 5.0 A Da(-1) and a solvent content of 75%. An interpretable electron-density map for the MAM K201A mutant crystal was produced using the molecular-replacement method.
• Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., Deas, T. S., Zhou, Y., Li, H., & Shi, P. Y. (2006). West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5.. Journal of virology, 80(17), 8362-70. doi:10.1128/jvi.00814-06
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  Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
• Li, H., Zhao, Y., Li, Z., Guo, Y., Li, Y., Vranken, S. V., & Eisele, L. E. (2005). Crystal structures of T cell receptor (beta) chains related to rheumatoid arthritis.. Protein science : a publication of the Protein Society, 14(12), 3025-38. doi:10.1110/ps.051748305
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  The crystal structures of the Vbeta17+ beta chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1(F104-->Y/C187-->S)) forms, respectively. These TCR beta chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 Vbeta17 closely resemble those of a Vbeta17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three Vbeta17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the Vbeta regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 Vbeta17, implying that there is considerable variability in the structures of very similar beta chains. Structural alignments also reveal a considerable variation in the Vbeta-Cbeta associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the Vbeta domains of known TCR structures indicate that there are significant similarities among Vbeta regions that are MAM-reactive, whereas there appear to be significant structural differences among those Vbeta regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.
• Li, Z., Li, H., Devasahayam, G., Chaturvedi, V., Hanes, S. D., Roey, P. V., & Gemmill, T. R. (2005). The structure of the Candida albicans Ess1 prolyl isomerase reveals a well-ordered linker that restricts domain mobility.. Biochemistry, 44(16), 6180-9. doi:10.1021/bi050115l
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  Ess1 is a peptidyl-prolyl cis/trans isomerase (PPIase) that binds to the carboxy-terminal domain (CTD) of RNA polymerase II. Ess1 is thought to function by inducing conformational changes in the CTD that control the assembly of cofactor complexes on the transcription unit. Ess1 (also called Pin1) is highly conserved throughout the eukaryotic kingdom and is required for growth in some species, including the human fungal pathogen Candida albicans. Here we report the crystal structure of the C. albicansEss1 protein, determined at 1.6 A resolution. The structure reveals two domains, the WW and the isomerase domain, that have conformations essentially identical to those of human Pin1. However, the linker region that joins the two domains is quite different. In human Pin1, this linker is short and flexible, and part of it is unstructured. In contrast, the fungal Ess1 linker is highly ordered and contains a long alpha-helix. This structure results in a rigid juxtaposition of the WW and isomerase domains, in an orientation that is distinct from that observed in Pin1, and that eliminates a hydrophobic pocket between the domains that was implicated as the main substrate recognition site. These differences suggest distinct modes of interaction with long substrate molecules, such as the CTD of RNA polymerase II. We also show that C. albicans ess1(-)() mutants are attenuated for in vivo survival in mice. Together, these results suggest that CaEss1 might constitute a useful antifungal drug target, and that structural differences between the fungal and human enzymes could be exploited for drug design.
• Zhao, Y., Li, Z., Drozd, S. J., Guo, Y., Mourad, W., & Li, H. (2004). Crystal structure of Mycoplasma arthritidis mitogen complexed with HLA-DR1 reveals a novel superantigen fold and a dimerized superantigen-MHC complex.. Structure (London, England : 1993), 12(2), 277-88. doi:10.1016/j.str.2004.01.008
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  Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the crystal structure of MAM complexed with a major histocompatibility complex (MHC) antigen, HLA-DR1, loaded with haemagglutinin peptide 306-318 (HA). The structure reveals that MAM has a novel fold composed of two alpha-helical domains. This fold is entirely different from that of the pyrogenic superantigens, consisting of a beta-grasped motif and a beta barrel. In the complex, the N-terminal domain of MAM binds orthogonally to the MHC alpha1 domain and the bound HA peptide, and to a lesser extent to the MHC beta1 domain. Two MAM molecules form an asymmetric dimer and cross-link two MHC antigens to form a plausible, dimerized MAM-MHC complex. These data provide the first crystallographic evidence that superantigens can dimerize MHC molecules. Based on our structure, a model of the TCR2MAM2MHC2 complex is proposed.
• Zhao, Y., Li, Z., Drozd, S., Guo, Y., Mourad, W., & Li, H. (2004). Crystal Structure of Mitogen Complexed with HLA-DR1 Reveals a Novel Superantigen Fold and a Dimerized Superantigen-MHC Complex. Structure, 12(2), 277-288. doi:10.1016/s0969-2126(04)00020-6
• Zhao, Y., Li, Z., Guo, Y., Li, H., Stack, R. F., Hauer, C. R., & Drozd, S. J. (2004). Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen.. Acta crystallographica. Section D, Biological crystallography, 60(Pt 2), 353-6. doi:10.1107/s090744490302763x
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  Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A. Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.
• Li, Y., Li, H., Yang, F., Smith-gill, S. J., & Mariuzza, R. A. (2003). X-ray snapshots of the maturation of an antibody response to a protein antigen.. Nature structural biology, 10(6), 482-8. doi:10.1038/nsb930
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  The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to protein provides insights into the evolution of high affinity in other protein-protein interfaces.
• Lu, L., Lamm, M. E., Li, H., Corthesy, B., & Zhang, J. (2003). The human polymeric immunoglobulin receptor binds to Streptococcus pneumoniae via domains 3 and 4.. The Journal of biological chemistry, 278(48), 48178-87. doi:10.1074/jbc.m306906200
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  Streptococcus pneumoniae (the pneumococcus) is a major cause of bacterial pneumonia, middle ear infection (otitis media), sepsis, and meningitis. Our previous study demonstrated that the choline-binding protein A (CbpA) of S. pneumoniae binds to the human polymeric immunoglobulin receptor (pIgR) and enhances pneumococcal adhesion to and invasion of cultured epithelial cells. In this study, we sought to determine the CbpA-binding motif on pIgR by deletional analysis. The extra-cellular portion of pIgR consists of five Ig-like domains (D1-D5), each of which contains 104-114 amino acids and two disulfide bonds. Deletional analysis of human pIgR revealed that the lack of either D3 or D4 resulted in the loss of CbpA binding, whereas complete deletions of domains D1, D2, and D5 had undetectable impacts. Subsequent analysis showed that domains D3 and D4 together were necessary and sufficient for the ligand-binding activity. Furthermore, CbpA binding of pIgR did not appear to require Ca2+ or Mg2+. Finally, treating pIgR with a reducing agent abolished CbpA binding, suggesting that disulfide bonding is required for the formation of CbpA-binding motif(s). These results strongly suggest a conformational CbpA-binding motif(s) in the D3/D4 region of human pIgR, which is functionally separated from the IgA-binding site(s).
• Wang, H., Lee, H. K., Bukowski, J. F., Li, H., Mariuzza, R. A., Chen, Z. W., Morita, C. T., & Nam, K. H. (2003). Conservation of nonpeptide antigen recognition by rhesus monkey V gamma 2V delta 2 T cells.. Journal of immunology (Baltimore, Md. : 1950), 170(7), 3696-706. doi:10.4049/jimmunol.170.7.3696
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  We have previously found that monkey Vgamma2Vdelta2(+) T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guérin infections. We have now analyzed rhesus monkey gammadelta T cell responses to nonpeptide Ags and superantigens. Like human Vgamma2Vdelta2(+) T cells, rhesus monkey gammadelta T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags. Responsiveness was limited to gammadelta T cells expressing Vgamma2Vdelta2 TCRs. Rhesus monkey Vgamma2Vdelta2(+) T cells also responded to the superantigen, staphyloccocal enterotoxin A. Sequencing of the rhesus monkey Vgamma2Vdelta2 TCR revealed a strong sequence homology to human Vgamma2Vdelta2 TCR that preserves important sequence motifs. Moreover, chimeric TCRs that pair human Vgamma2 with monkey Vdelta2 and monkey Vgamma2 with human Vdelta2 retain reactivity to nonpeptide Ags and B cell lymphomas. A molecular model of the rhesus monkey Vgamma2Vdelta2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vgamma2Vdelta2 TCR and preserves the topology of the complementarity-determining region loops. Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human gammadelta T cell Ag responses.
• Wong, S. J., Boyle, R. H., Demarest, V. L., Woodmansee, A. N., Kramer, L. D., Li, H., Koski, R. A., Fikrig, E., Martin, D. A., Shi, P. Y., & Drebot, M. A. (2003). Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination.. Journal of clinical microbiology, 41(9), 4217-23. doi:10.1128/jcm.41.9.4217-4223.2003
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  West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.
• Etongue-mayer, P., Langlois, M., Ouellette, M., Li, H., Younes, S., Al-daccak, R., & Mourad, W. (2002). Involvement of zinc in the binding of Mycoplasma arthritidis-derived mitogen to the proximity of the HLA-DR binding groove regardless of histidine 81 of the beta chain.. European journal of immunology, 32(1), 50-8. doi:10.1002/1521-4141(200201)32:1<50::aid-immu50>3.0.co;2-a
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  Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.
• Li, H., Li, Z., Takegawa, K., & Roey, P. V. (2002). Structural basis for the transglycosylation activity of endo-β-N-acetylglucosaminidase A. Acta Crystallographica Section A, 58(s1), 106-106. doi:10.1107/s0108767302089250
• Sundberg, E. J., Li, H., Llera, A. S., Mccormick, J. K., Tormo, J., Schlievert, P. M., Karjalainen, K., & Mariuzza, R. A. (2002). Structures of two streptococcal superantigens bound to TCR beta chains reveal diversity in the architecture of T cell signaling complexes.. Structure (London, England : 1993), 10(5), 687-99. doi:10.1016/s0969-2126(02)00759-1
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  Superantigens (SAGs) crosslink MHC class II and TCR molecules, resulting in an overstimulation of T cells associated with human disease. SAGs interact with several different surfaces on MHC molecules, necessitating the formation of multiple distinct MHC-SAG-TCR ternary signaling complexes. Variability in SAG-TCR binding modes could also contribute to the structural heterogeneity of SAG-dependent signaling complexes. We report crystal structures of the streptococcal SAGs SpeA and SpeC in complex with their corresponding TCR beta chain ligands that reveal distinct TCR binding modes. The SpeC-TCR beta chain complex structure, coupled with the recently determined SpeC-HLA-DR2a complex structure, provides a model for a novel T cell signaling complex that precludes direct TCR-MHC interactions. Thus, highly efficient T cell activation may be achieved through structurally diverse strategies of TCR ligation.
• Lee, H. K., Rich, R. R., Morita, C. T., Mariuzza, R. A., Li, H., Lamphear, J. G., & Fraser, J. D. (2001). Superantigen recognition by gammadelta T cells: SEA recognition site for human Vgamma2 T cell receptors.. Immunity, 14(3), 331-44. doi:10.1016/s1074-7613(01)00113-3
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  Human gammadelta T cells expressing the Vgamma2Vdelta2 antigen receptors recognize nonpeptide prenyl pyrophosphate and alkylamine antigens. We find that they also recognize staphylococcal enterotoxin A superantigens in a manner distinct from the recognition of nonpeptide antigens. Using chimeric and mutant toxins, SEA amino acid residues 20-27 were shown to be required for gammadelta TCR recognition of SEA. Residues at 200-207 that are critical for specific alphabeta TCR recognition of SEA do not affect gammadelta TCR recognition. SEA residues 20-27 are located in an area contiguous with the binding site of V beta chains. This study defines a superantigen recognition site for a gammadelta T cell receptor and demonstrates the differences between Vgamma2Vdelta2+ T cell recognition of superantigens and nonpeptide antigens.
• Li, Y., Li, H., Dimasi, N., Mccormick, J. K., Martin, R., Schuck, P., Schlievert, P. M., & Mariuzza, R. A. (2001). Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II.. Immunity, 14(1), 93-104. doi:10.1016/s1074-7613(01)00092-9
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  MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.
• Morita, C. T., Lee, H. K., Wang, H., Li, H., Mariuzza, R. A., & Tanaka, Y. (2001). Structural features of nonpeptide prenyl pyrophosphates that determine their antigenicity for human gamma delta T cells.. Journal of immunology (Baltimore, Md. : 1950), 167(1), 36-41. doi:10.4049/jimmunol.167.1.36
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  Human Vgamma2Vdelta2(+) T cells proliferate in vivo during many microbial infections. We have found that Vgamma2Vdelta2(+) T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vgamma2Vdelta2(+) clones and a transfectant responded similarly. Ag docking into a Vgamma2Vdelta2 TCR model reveals a potential binding site in germline regions of the Vgamma2Jgamma1.2 CDR3 and Vdelta2 CDR2 loops. Thus, Vgamma2Vdelta2(+) T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.
• He, X., Liu, X., Li, H., Wang, M., Zhang, Y., Zeng, Z., & Wang, D. (2000). Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm.. Science in China. Series C, Life sciences, 43(1), 39-46. doi:10.1007/bf02881716
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  BmK M4 is a neutral neurotoxin in the BmK toxin series. It is medially toxic and belongs to group III alpha-toxins. The purified sample was crystallized in rhombic space group P6(1). Using an X-ray diffraction technique, the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution. The model was refined. The final crystallographic R factor was 0.142 and the freeR factor was 0.173. The root mean square deviation is 0.001 5 nm for the bond length and 1.753 degrees for the bond angles. 64 water molecules were added to the asymmetric unit. The refined structure showed an unusual non-prolyl cis peptide bond at residue 10. The structure was compared with group II alpha-toxin BmK M8 (an acidic, weak toxin). The potential structural implications of the cis peptide bond were discussed.
• Li, Y., Li, H., Martin, R., & Mariuzza, R. A. (2000). Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins.. Journal of molecular biology, 304(2), 177-88. doi:10.1006/jmbi.2000.4198
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  Susceptibility to multiple sclerosis (MS) is associated with certain MHC class II haplotypes, in particular HLA-DR2. Two DR beta chains, DRB1*1501 and DRB5*0101, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP 84-102) to MBP-specific T cells from MS patients. We have determined the crystal structure of HLA-DR2a complexed with MBP 86-105 to 1.9 A resolution. A comparison of this structure with that of HLA-DR2b complexed with MBP 85-99, reported previously, reveals that the peptide register is shifted by three residues, such that the MBP peptide is bound in strikingly different conformations by the two MHC molecules. This shift in binding register is attributable to a large P1 pocket in DR2a, which accommodates Phe92, in conjunction with a relatively shallow P4 pocket, which is occupied by Ile95. In DR2b, by contrast, the small P1 pocket accommodates Val89, while the deep P4 pocket is filled by Phe92. In both complexes, however, the C-terminal half of the peptide is positioned higher in the binding groove than in other MHC class II/peptide structures. As a result of the register shift, different side-chains of the MBP peptide are displayed for interaction with T cell receptors in the DR2a and DR2b complexes. These results demonstrate that MHC molecules can impose different alignments and conformations on the same bound peptide as a consequence of topological differences in their peptide-binding sites, thereby creating distinct T cell epitopes.
• Li, Y., Li, H., Smith-gill, S. J., & Mariuzza, R. A. (2000). Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,).. Biochemistry, 39(21), 6296-309. doi:10.1021/bi000054l
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  Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small ( 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (
• Li, H. M., Zhang, Y., Zeng, Z. H., Yao, Z. P., Wang, D. C., & Feng, Y. M. (1999). Structure of an insulin dimer in an orthorhombic crystal: the structure analysis of a human insulin mutant (B9 Ser-->Glu).. Acta crystallographica. Section D, Biological crystallography, 55(Pt 9), 1524-32. doi:10.1107/s0907444999008562
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  The structure of human insulin mutant B9 (Ser-->Glu) was determined by an X-ray crystallographic method at 2.5 A resolution with an R factor of 0.165 under non-crystallographic restraints. The crystals were grown at low pH (
• Li, H. M., Zhao, T., Jin, L., Wang, M., Zhang, Y., & Wang, D. C. (1999). A series of bioactivity-variant neurotoxins from scorpion Buthus martensii Karsch: purification, crystallization and crystallographic analysis.. Acta crystallographica. Section D, Biological crystallography, 55(Pt 1), 341-4. doi:10.1107/s0907444998006593
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  Three bioactivity-variant neurotoxins, BmK M1, M4 and M8, have been purified from Chinese scorpion BmK venom. They possess distinct toxic activities against mice in vivo. These proteins also have different electrostatic properties. The relative toxicities for BmK M1, M4 and M8 are 13.3:2.5:1 which, surprisingly, correspond to their respective pI values ranging from basic to acidic 9.01, 7.53 and 5.30, respectively. They have been crystallized in different crystal forms as orthorhombic, hexagonal and monoclinic, respectively. These crystals can diffract to 1.2 (BmK M1), 1.3 (BmK M4) and 1.8 A (BmK M8) resolution and have been used in data collection. These toxins produced by natural mutagenesis or gene divergence should represent functionally distinct states, thereby forming a valuable system for studying structure-function relationships. The unusual relatively acidic component that first appeared in this series also provides a new concept for a more comprehensive understanding of scorpion neurotoxins.
• Li, H., Malchiodi, E. L., Mariuzza, R. A., & Llera, A. S. (1999). The structural basis of T cell activation by superantigens.. Annual review of immunology, 17(1), 435-66. doi:10.1146/annurev.immunol.17.1.435
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  Superantigens (SAGs) are a class of immunostimulatory and disease-causing proteins of bacterial or viral origin with the ability to activate large fractions (5-20%) of the T cell population. Activation requires simultaneous interaction of the SAG with the V beta domain of the T cell receptor (TCR) and with major histocompatibility complex (MHC) class II molecules on the surface of an antigen-presenting cell. Recent advances in knowledge of the three-dimensional structure of bacterial SAGs, and of their complexes with MHC class II molecules and the TCR beta chain, provide a framework for understanding the molecular basis of T cell activation by these potent mitogens. These structures along with those of TCR-peptide/MHC complexes reveal how SAGs circumvent the normal mechanism for T cell activation by peptide/MHC and how they stimulate T cells expressing TCR beta chains from a number of different families, resulting in polyclonal T cell activation. The crystal structures also provide insights into the basis for the specificity of different SAGs for particular TCR beta chains, and for the observed influence of the TCR alpha chain on SAG reactivity. These studies open the way to the design of SAG variants with altered binding properties for TCR and MHC for use as tools in dissecting structure-activity relationships in this system.
• Dall'acqua, W., Goldman, E. R., Lin, W., Teng, C., Tsuchiya, D., Li, H., Ysern, X., Braden, B. C., Li, Y., Smith-gill, S. J., & Mariuzza, R. A. (1998). A mutational analysis of binding interactions in an antigen-antibody protein-protein complex.. Biochemistry, 37(22), 7981-91. doi:10.1021/bi980148j
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  Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine. Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively. The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.
• Jin, L., Li, H., Hu, Z., Zeng, Z., & Wang, D. (1998). Crystal structure of pokeweed antiviral protein from seeds ofPhytolacca americana at 0.25 nm.. Science in China. Series C, Life sciences, 41(4), 413-8. doi:10.1007/bf02882742
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  Crystals of pokeweed antiviral protein (PAP) from seeds ofPhytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33 degrees C). The crystal structure was solved by use of molecular replacement method and refied by use of molecular dynamic method at 0.25 nm to anR factor of 18.15% with standard deviations from standard geometry of 0.001 6 nm and 2.04 for bond lengths and bond angles, respectively. Comparison with two other PAPS revealed, near the active center, a sequence- and structure-variable region, consisting of the loop connecting the fifth beta-strand with the second alpha-helix and including a proposed active residue, suggesting this loop probably to be related to difference in activity.
• Leder, L., Lavoie, P. M., Lebedeva, M. I., Li, H., Bohach, G. A., Gahr, P. J., Schlievert, P. M., Karjalainen, K., Mariuzza, R. A., Sekaly, R. P., & Llera, A. S. (1998). A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.. The Journal of experimental medicine, 187(6), 823-33. doi:10.1084/jem.187.6.823
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  The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.
• Li, H. M., Hu, Z., Zeng, Z. H., & Wang, D. C. (1998). Crystallization and preliminary crystallographic analyses of pokeweed antiviral protein from seeds.. Acta crystallographica. Section D, Biological crystallography, 54(Pt 1), 137-9. doi:10.1107/s0907444997010639
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  Pokeweed antiviral protein from seeds (PAP-S) is a ribosome inactivating protein which has lowest toxicity and highest inhibition activity as opposed to other pokeweed antiviral proteins and its three potential glycosylation sites (10, 44, 255) were shown to bind to N-acetylglucosamine. Good quality crystals of PAP-S were grown at high protein concentration (100 mg ml-1) and high temperature (306 K). The crystals have space group I222 and cell parameters a = 78.7, b = 85.2 and c = 93.0 A. An X-ray diffraction data set with resolution up to 1.8 A was collected. This high-resolution data will help to locate the sugars bound to the protein and provide accurate structural data for understanding structure-function relationships of PAP-S.
• Li, H., Mariuzza, R. A., & Llera, A. S. (1998). Structure-function studies of T-cell receptor-superantigen interactions.. Immunological reviews, 163(1), 177-86. doi:10.1111/j.1600-065x.1998.tb01196.x
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  Superantigens (SAGs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the V beta domain of the T-cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR beta chain (mouse V beta 8.2-J beta 2.1-C beta 1) and the SAG staphylococcal enterotoxin C3 (SEC3) has been recently determined. The complementarity-determining region 2 (CDR2) of the beta chain and, to lesser extents, CDR1 and hypervariable region 4 (HV4) bind in a cleft between the small and large domains of the SAG. A model of the TCR-SAG-peptide/MHC complex constructed from available crystal structures reveals how the SAG acts as a wedge between the TCR and MHC, thereby displacing the antigenic peptide away from the TCR and circumventing the normal mechanism for T-cell activation by peptide/MHC. To evaluate the actual contribution of individual SAG residues to stabilizing the V beta C beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCB and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 mutants to a soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. We show that there is direct correlation between affinity and ability to stimulate T cells, with SAGs having the highest affinity for the TCR being the most biologically active. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the V beta C beta-SEC3 complex are strictly conserved among enterotoxins reactive with mouse V beta 8.2, thereby explaining why SAGs having other residues at these positions show different V beta-binding specificities.
• Li, H., Natarajan, K., Malchiodi, E. L., Margulies, D. H., & Mariuzza, R. A. (1998). Three-dimensional structure of H-2Dd complexed with an immunodominant peptide from human immunodeficiency virus envelope glycoprotein 120.. Journal of molecular biology, 283(1), 179-91. doi:10.1006/jmbi.1998.2091
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  The crystal structure of the mouse major histocompatibility complex (MHC) class I molecule H-2Dd with an immunodominant peptide, designated P18-I10 (RGPGRAFVTI), from human immunodeficiency virus envelope glycoprotein 120 was determined at 3.2 A resolution. A novel orientation of the alpha3 domain of Dd relative to the alpha1/alpha2 domains results in significantly fewer contacts between alpha3 and beta2-microglobulin compared with other MHC class I proteins. Four out of ten peptide residues (P2 Gly, P3 Pro, P5 Arg and P10 Ile) are nearly completely buried in the Dd binding groove. This is consistent with previous findings that Dd exploits a four-residue binding motif comprising a glycine at P2, a proline at P3, a positively charged residue at P5, and a C-terminal hydrophobic residue at P9 or P10. The side-chain of P5 Arg is directed toward the floor of the predominantly hydrophobic binding groove where it forms two salt bridges and one hydrogen bond with Dd residue Asp77. The selection of glycine at P2 appears to be due to a narrowing of the B pocket, relative to that of other class I molecules, caused by Arg66 whose side-chain folds down into the binding cleft. Residue P3 Pro of P18-I10 occupies part of pocket D, which in Dd is partially split by a prominent hydrophobic ridge in the floor of the binding groove formed by Trp97 and Trp114. Residues P6 through P9 form a solvent-exposed bulge, with P7 Phe protruding the most from the binding groove and thereby probably constituting a major site of interaction with T cell receptors. A comparison of H-2Dd/P18-I10 with other MHC class I/peptide complexes of known structure provides insights into the possible basis for the specificity of the natural killer cell receptor Ly-49A for several related class I molecules.
• Li, H., Tsuchiya, D., Leder, L., Ysern, X., Schlievert, P. M., Karjalainen, K., Mariuzza, R. A., & Llera, A. S. (1998). Three-dimensional structure of the complex between a T cell receptor beta chain and the superantigen staphylococcal enterotoxin B.. Immunity, 9(6), 807-16. doi:10.1016/s1074-7613(00)80646-9
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  Superantigens (SAGs) are a class of immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the V beta domain of the T cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR beta chain (mouse V beta8.2) and the SAG staphylococcal enterotoxin B (SEB) at 2.4 A resolution reveals why SEB recognizes only certain V beta families, as well as why only certain SAGs bind mouse V beta8.2. Models of the TCR-SEB-peptide/MHC class II complex indicate that V alpha interacts with the MHC beta chain in the TCR-SAG-MHC complex. The extent of the interaction is variable and is largely determined by the geometry of V alpha/V beta domain association. This variability can account for the preferential expression of certain V alpha regions among T cells reactive with SEB.
• Mariuzza, R. A., Llera, A. S., Li, H., Lebedeva, M. I., Fields, B. A., & Brenner, M. B. (1998). Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.. Nature, 391(6666), 502-6. doi:10.1038/35172
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  Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of alpha and beta, or gamma and delta, polypeptide chains with variable (V) and constant (C) regions. In contrast to alphabeta TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), gammadelta TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certain gammadelta T cells. These studies indicate that antigen recognition by gammadelta TCRs may be fundamentally different from that by alphabeta TCRs. The three-dimensional structures of several alphabeta TCRs and TCR fragments, and their complexes with peptide-MHC or superantigens, have been determined. Here we report the crystal structure of the Vdelta domain of a human gammadelta TCR at 1.9 A resolution. A comparison with antibody and alphabeta TCR V domains reveals that the framework structure of Vdelta more closely resembles that of VH than of Valpha, Vbeta or VL (where H and L refer to heavy and light chains), whereas the relative positions and conformations of its complementarity-determining regions (CDRs) share features of both Valpha and VH. These results provide the first direct evidence that gammadelta TCRs are structurally distinct from alphabeta TCRs and, together with the observation that the CDR3 length distribution of TCR delta chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by gammadelta TCRs may resemble antigen recognition by antibodies.
• Ward, E. S., Mariuzza, R. A., Li, H., & Lebedeva, M. I. (1997). Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association.. Journal of molecular biology, 269(3), 385-94. doi:10.1006/jmbi.1997.1047
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  The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.
• Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., & Mariuzza, R. A. (1996). Crystal structure of a T-cell receptor beta-chain complexed with a superantigen.. Nature, 384(6605), 188-92. doi:10.1038/384188a0
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  Superantigens (SAgs) are viral or bacterial proteins that act as potent T-cell stimulants and have been implicated in a number of human diseases, including toxic shock syndrome, diabetes mellitus and multiple sclerosis. The interaction of SAgs with the T-cell receptor (TCR) and major histocompatibility complex (MHC) proteins results in the stimulation of a disproportionately large fraction of the T-cell population. We report here the crystal structures of the beta-chain of a TCR complexed with the Staphylococcus aureus enterotoxins C2 and C3 (SEC2, SEC3). These enterotoxins, which cause both toxic shock and food poisoning, bind in an identical way to the TCR beta-chain. The complementarity-determining region 2 (CDR2) of the beta-chain and, to lesser extents, CDR1 and hypervariable region 4 (HV4), bind in a cleft between the two domains of the SAgs. Thus, there is considerable overlap between the SAg-binding site and the peptide/MHC-binding sites of the TCR. A model of a TCR-SAg-MHC complex constructed from the crystal structures of (1) the beta-chain-SEC3 complex, (2) a complex between staphylococcal enterotoxin B (SEB) and an MHC molecule, and (3) a TCR V(alpha) domain, reveals that the SAg acts as a wedge between the TCR and MHC to displace the antigenic peptide away from the TCR combining site. In this way, the SAg is able to circumvent the normal mechanism for T-cell activation by specific peptide/MHC complexes.
• Li, H. M., Jin, L., Zeng, Z. H., Wang, D. C., & Hu, R. Q. (1996). Crystal structure of an acidic neurotoxin from scorpion Buthus martensii Karsch at 1.85 A resolution.. Journal of molecular biology, 261(3), 415-31. doi:10.1006/jmbi.1996.0473
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  The crystal structure of an acidic scorpion neurotoxin, BmK M8, purified from Chinese scorpion Buthus martensii Karsch (BmK), has been determined by the molecular replacement method. It is the first structure of an acidic alpha-scorpion neurotoxin reported so far. The crystals adopt a symmetry of space group P2(1) and contain one molecule per asymmetric unit. The structure has been refined to an R factor of 18.1% using reflection data in the range of 8 to 1.85 A resolution, with standard deviations from ideal geometry of 0.017 A and 2.43 degrees for bond length and angle, respectively. The 12 residues at the C terminus with unknown sequence were determined by crystallographic refinement. The refined model shows that the structural core, consisting of a motif beta alpha beta beta, is similar to that of toxin II from Androctonus australis Hector (AaH II) or Variant 3 from Centruroides sculpturatus Ewing (CsE V3). The three conformationally variable loops protruding from this structural core are different from that of AaH II, and especially from that of CsE V3. Compared with the most potent and basic alpha-toxin AaH II, the BmK M8 is a relatively inactive toxin (1100 times less active than AaH II) with an unusually low isoelectric point (pI 5.3). Sequence alignment of the two toxins shows a difference of 26 residues (40.6%). Among them four basic or neutral residues in AaH II, namely Val10, Lys28, Val55 and Gly59, are changed to acidic glutamate in BmK M8. The residues Glu10, Glu28 and Glu55 of BmK M8 are located on a surface (Face B), opposite the "conserved hydrophobic surface" (Face A). The latter is a functionally important area proposed by Fontecilla-Camps et al. Our observations suggest that in addition to Face A, Face B may also be involved in the biological activity of scorpion toxins. The structure of BmK M8 shows an evident conformational change of the alpha-amino group at the N terminus and a deorganization of Arg2 caused by the mutation D53A. These structural changes may also be responsible for the weak toxicity of BmK M8. In association with the information from chemical modifications, a multisite binding mode for toxin-receptor interaction and three "toxic regions" in relevance to the binding process, including Face A, Face B and Site C, are proposed. Face A, mainly consisting of Tyr5, 35, 47, the alpha-amino group, Arg2 and Asp3, may be more essential for the binding. Face B, mainly comprising conserved residues Tyr14, 21, Lys28 and Val55, may contribute to the high efficacy of the binding process and substitutions by acidic residues in this area could strongly weaken the toxic activity. Site C, formed by Lys58 and Arg62 at the C terminus and Arg41 and Tyr42 from loop 38-44, may be involved in binding site specificity.
• Li, H., Jin, L., Wang, M., Zhang, Y., Wang, D., & Zeng, Z. (1996).

  Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthus martensii Karsch at 0.25 nm resolution.

  . Science in China. Series C, Life sciences, 39(4), 373-84.
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  The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25 nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5-0.35 nm. The crystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.0017 nm and 2.24 degrees, respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of alpha-helix with two and a half turns (residues 19-28) and a three-stranded antiparallel beta-sheet (residues 2-4, 32-37, 45-51). In addition, three loops protruded from the structural core. The general folding properties of BmK M8 molecule were described; a common structure motif which may appear in all scorpion neurotoxins was identified. The conserved aromatic residues and charged residues were found to be distributed on two roughly opposite surfaces of the molecule. The relationship between these two faces and receptor-binding sites are also discussed.
• Li, H., Lebedeva, M. I., Ward, E. S., & Mariuzza, R. A. (1996). Crystal structure of a CDR3 mutant of a T cell antigen receptor Va domain. Acta Crystallographica Section A, 52(a1), 221-221. doi:10.1107/s0108767396090514
• Mariuzza, R. A., Li, H., Malchiodi, E. L., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainan, K., & Fields, B. A. (1996). Structure of a superantigen T-cell receptor chain complex. Acta Crystallographica Section A, 52(a1), 581-581. doi:10.1107/s0108767396076362
• Wang, D. C., Li, H. M., Jin, L., & Zeng, Z. H. (1996). Crystal structure of an acidic neurotoxin from scorpion Buthus martensii Karsch at 1.85 Å resolution. Acta Crystallographica Section A, 52(a1), 247-247. doi:10.1107/s0108767396089544
• Jin, L., Li, H., Wang, M., Wang, D., & Zeng, Z. H. (1993). Crystallographic study on a series of neurotoxins with different toxicities from Chinese scorpions. Acta Crystallographica Section A, 49(s1), c118-c118. doi:10.1107/s0108767378096610