- Professor, Chemistry and Biochemistry
- Professor, Chemistry and Biochemistry-Sci
- Professor, BIO5 Institute
My laboratory uses X-ray crystallography , NMR spectroscopy and small angle X-ray scattering (SAXS) in combination with biochemistry and genetics to understand the molecular basis of signaling, with a particular interest in understanding how bacterial signaling proteins regulate biolfim formation and antibiotic resistance, how targeting proteins direct the activity of ser/thr phosphatases and how dual specificity and tyrosine phosphatases regulate MAPK function.
- Ph.D. Chemistry
- Princeton University, Princeton, New Jersey, United States
- Domain Motions in Actin and their Implications for F-actin Modeling.
- B.A. Applied Mathematics
- University of Arizona, Tucson, Arizona, United States
- B.S. Biochemistry
- University of Arizona, Tucson, Arizona, United States
- Professor of Chemistry and Biochemistry, University of Arizona, College of Medicine (2017 - Ongoing)
- Professor of Chemistry and Biochemistry, University of Arizona, College of Science (2017 - Ongoing)
- Professor of Biology, Brown University, Providence, Rhode Island (2015 - 2017)
- Associate Professor of Biology, tenured, Brown University, Providence, Rhode Island (2011 - 2015)
- Assistant Professor of Biology, tenure-track, Brown University, Providence, Rhode Island (2005 - 2011)
- Assistant Professor, research, Brown University, Providence, Rhode Island (2004 - 2005)
- Core Project Leader, Crystallomics Core, The Scripps Research Institute (2003 - 2004)
- Post-doctoral Research Scientist, The Scripps Research Institute (2000 - 2003)
- Harold W. Dodds Honorific Graduate Fellowship
- Princeton University, Fall 1997
- National Science Foundation Graduate Fellowship
- NSF, Fall 1994
- Phi Beta Kappa
- Phi Beta Kappa, Fall 1993
- Barry M. Goldwater Undergraduate Research Scholar
- Fall 1992
- Regent’s Academic Achievement Scholarship (4 years)
- Arizona Board of Regents, Fall 1988
- Donna B. Cosulich Fellow
- Spring 2017
- NSF-CAREER awardee
- NSF, Spring 2016
- Eighteenth Annual Gehrenbeck Lecturer
- Rhode Island College, Fall 2013
- Ruth L Kirschstein National Research Service Award (NRSA), NIH (F32NS011146)
- NIH, Summer 2009
- American Cancer Society Research Scholar
- American Cancer Society, Summer 2008
No activities entered.
DissertationBIOC 920 (Spring 2018)
Exchange Chemical InfoCHEM 695B (Spring 2018)
Fndtns in BiochemistryBIOC 384 (Spring 2018)
Honors Independent StudyNSCS 299H (Spring 2018)
Introduction to ResearchMCB 795A (Spring 2018)
ResearchBIOC 900 (Spring 2018)
Senior CapstoneBIOC 498 (Spring 2018)
Introduction to ResearchMCB 795A (Fall 2017)
Metabolic BiochemistryBIOC 385 (Fall 2017)
ResearchCHEM 900 (Fall 2017)
Senior CapstoneBIOC 498 (Fall 2017)
Fndtns in BiochemistryBIOC 384 (Spring 2017)
- Peti, W., Page, R., Boura, E., & Różycki, B. (2018). Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System. Methods in molecular biology (Clifton, N.J.), 1688, 375-389.More infoThe integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.
- Choy, M. S., Li, Y., Machado, L., Kunze, M., Connors, C. R., Wei, X., Lindorff-Larsen, K., Page, R., & Peti, W. (2017). Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery. MOLECULAR CELL, 65(4), 644-+.
- Choy, M. S., Swingle, M., D'Arcy, B., Abney, K., Rusin, S. F., Kettenbach, A. N., Page, R., Honkanen, R. E., & Peti, W. (2017). PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors. Journal of the American Chemical Society, 139(49), 17703-17706.More infoSelective inhibitors for each serine/threonine phosphatase (PPP) are essential to investigate the biological actions of PPPs and to guide drug development. Biologically diverse organisms (e.g., cyanobacteria, dinoflagellates, beetles) produce structurally distinct toxins that are catalytic inhibitors of PPPs. However, most toxins exhibit little selectivity, typically inhibiting multiple family members with similar potencies. Thus, the use of these toxins as chemical tools to study the relationship between individual PPPs and their biological substrates, and how disruptions in these relationships contributes to human disease, is severely limited. Here, we show that tautomycetin (TTN) is highly selective for a single PPP, protein phosphatase 1 (PP1/PPP1C). Our structure of the PP1:TTN complex reveals that PP1 selectivity is defined by a covalent bond between TTN and a PP1-specific cysteine residue, Cys127. Together, these data provide key molecular insights needed for the development of novel probes targeting single PPPs, especially PP1.
- Machado, L. E., Critton, D. A., Page, R., & Peti, W. (2017). Redox Regulation of a Gain-of-Function Mutation (N308D) in SHP2 Noonan Syndrome. ACS omega, 2(11), 8313-8318.More infoSHP2 (Src homology 2 domain-containing protein tyrosine phosphatase 2; PTPN11) is a ubiquitous multidomain, nonreceptor protein tyrosine phosphatase (PTP) that plays an important role in diseases such as cancer, diabetes, and Noonan syndrome (NS). NS is one of the most common genetic disorders associated with congenital heart disease, and approximately half of the patients with Noonan syndrome have gain-of-function mutations in SHP2. One of the most common NS mutations is N308D. The activity of SHP2, like that of most PTPs, is reversibly inactivated by reactive oxygen species (ROS). However, the molecular basis of this inactivation and the consequences of NS-related mutations in PTPN11 on ROS-mediated inhibition are poorly understood. Here, we investigated the mechanistic and structural details of the reversible oxidation of the NS variant SHP2. We show that SHP2is more sensitive to oxidation when compared with wild-type SHP2. We also show that although the SHP2catalytic domain can be reactivated by dithiothreitol as effectively as the wild-type, full-length SHP2is only poorly reactivated by comparison. To understand the mechanism of oxidation at a molecular level, we determined the crystal structure of oxidized SHP2. The structure shows that the catalytic Cys459 residue forms a disulfide bond with Cys367, which confirms that Cys367 functions as the "backdoor" cysteine in SHP2. Together, our data suggest that the reversible oxidation of SHP2 contributes negligibly, if at all, to the symptoms associated with NS.
- Machado, L. E., Shen, T. L., Page, R., & Peti, W. (2017). The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation. The Journal of biological chemistry, 292(21), 8786-8796.More infoThe kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS),HO, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their HOoxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.
- Zhang, R., Lord, D. M., Bajaj, R., Peti, W., Page, R., & Sello, J. K. (2017). A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor. Nucleic acids research.More infoIn Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the β-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 μM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.
- Chen, E., Choy, M. S., Petrényi, K., Kónya, Z., Erdődi, F., Dombrádi, V., Peti, W., & Page, R. (2016). Molecular Insights into the Fungus-Specific Serine/Threonine Protein Phosphatase Z1 in Candida albicans. mBio, 7(4).More infoThe opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae]). Unexpectedly, we also discovered that, despite its 66% sequence identity to PP1α, the catalytic domain of PPZ1 contains novel structural elements that are not present in PP1α. We then used activity and pulldown assays to show that these structural differences block a large subset of PP1/GLC7 regulatory proteins from effectively binding PPZ1, demonstrating that PPZ1 does not compete with GLC7 for its regulatory proteins. Equally important, these unique structural elements provide new pockets suitable for the development of PPZ1-specific inhibitors. Together, these studies not only reveal why PPZ1 does not negatively impact GLC7 activity in vivo but also demonstrate that the family of fungus-specific phosphatases-especially PPZ1 from C. albicans-are highly suitable targets for the development of novel drugs that specifically target C. albicans without cross-reacting with human phosphatases.
- Kumar, G. S., Gokhan, E., De, M. S., Bonen, M., Vagnarelli, P., Peti, W., & Page, R. (2016). The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism. ELIFE, 5.
- Page, R., & Peti, W. (2016). Toxin-antitoxin systems in bacterial growth arrest and persistence. NATURE CHEMICAL BIOLOGY, 12(4), 208-214.
- Peti, W., & Page, R. (2016). NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases. Methods in molecular biology (Clifton, N.J.), 1447, 181-96.More infoNMR spectroscopy and other solution methods are increasingly being used to obtain novel insights into the mechanisms by which MAPK regulatory proteins bind and direct the activity of MAPKs. Here, we describe how interactions between the MAPK p38α and its regulatory proteins are studied using NMR spectroscopy, isothermal titration calorimetry, and small angle X-ray scattering (SAXS).
- Sheftic, S. R., Page, R., & Peti, W. (2016). Investigating the human Calcineurin Interaction Network using the pi Phi LxVP SLiM. SCIENTIFIC REPORTS, 6.
- Wang, X., Bajaj, R., Bollen, M., Peti, W., & Page, R. (2016). Expanding the PP2A Interactome by Defining a B56-Specific SLiM. STRUCTURE, 24(12), 2174-2181.
- Choy, M. S., Yusoff, P., Lee, I. C., Newton, J. C., Goh, C. W., Page, R., Shenolikar, S., & Peti, W. (2015). Structural and Functional Analysis of the GADD34:PP1 eIF2 alpha Phosphatase. CELL REPORTS, 11(12), 1885-1891.
- Krishnan, N., Krishnan, K., Connors, C. R., Choy, M. S., Page, R., Peti, W., Van, A. L., Shea, S. D., & Tonks, N. K. (2015). PTP1B inhibition suggests a therapeutic strategy for Rett syndrome. JOURNAL OF CLINICAL INVESTIGATION, 125(8), 3163-3177.
- Kwan, B. W., Lord, D. M., Peti, W., Page, R., Benedik, M. J., & Wood, T. K. (2015). The MqsR/MqsA toxin/antitoxin system protects Escherichia coli during bile acid stress. Environmental microbiology, 17(9), 3168-81.More infoToxin/antitoxin (TA) systems are ubiquitous within bacterial genomes, and the mechanisms of many TA systems are well characterized. As such, several roles for TA systems have been proposed, such as phage inhibition, gene regulation and persister cell formation. However, the significance of these roles is nebulous due to the subtle influence from individual TA systems. For example, a single TA system has only a minor contribution to persister cell formation. Hence, there is a lack of defining physiological roles for individual TA systems. In this study, phenotype assays were used to determine that the MqsR/MqsA type II TA system of Escherichia coli is important for cell growth and tolerance during stress from the bile salt deoxycholate. Using transcriptomics and purified MqsR, we determined that endoribonuclease toxin MqsR degrades YgiS mRNA, which encodes a periplasmic protein that promotes deoxycholate uptake and reduces tolerance to deoxycholate exposure. The importance of reducing YgiS mRNA by MqsR is evidenced by improved growth, reduced cell death and reduced membrane damage when cells without ygiS are stressed with deoxycholate. Therefore, we propose that MqsR/MqsA is physiologically important for E. coli to thrive in the gallbladder and upper intestinal tract, where high bile concentrations are prominent.
- Peti, W., & Page, R. (2015). Strategies to make protein serine/threonine (PP1, calcineurin) and tyrosine phosphatases (PTP1B) druggable: Achieving specificity by targeting substrate and regulatory protein interaction sites. BIOORGANIC & MEDICINAL CHEMISTRY, 23(12), 2781-2785.
- Choy, M. S., Hieke, M., Kumar, G. S., Lewis, G. R., Gonzalez-DeWhitt, K. R., Kessler, R. P., Stein, B. J., Hessenberger, M., Nairn, A. C., Peti, W., & Page, R. (2014). Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 111(11), 4097-4102.
- Francis, D. M., Koveal, D., Tortajada, A., Page, R., & Peti, W. (2014). Interaction of Kinase-Interaction-Motif Protein Tyrosine Phosphatases with the Mitogen-Activated Protein Kinase ERK2. PLOS ONE, 9(3).
- Francis, D. M., Page, R., & Peti, W. (2014). Sequence-specific backbone ¹H, ¹³C and ¹⁵N assignments of the 34 kDa catalytic domain of PTPN5 (STEP). Biomolecular NMR assignments, 8(1), 185-8.More infoPTPN5 is a protein tyrosine phosphatase that plays an integral role in regulating excitatory postsynaptic activity. The sequence-specific backbone assignments of the murine PTPN5 catalytic domain have been determined based on triple-resonance experiments using uniformly [(2)H,(13)C,(15)N]-labeled protein.
- Krishnan, N., Koveal, D., Miller, D. H., Xue, B., Akshinthala, S. D., Kragelj, J., Jensen, M. R., Gauss, C., Page, R., Blackledge, M., Muthuswamy, S. K., Peti, W., & Tonks, N. K. (2014). Targeting the disordered C terminus of PTP1B with an allosteric inhibitor. NATURE CHEMICAL BIOLOGY, 10(7), 558-566.
- Lord, D. M., Baran, A. U., Wood, T. K., Peti, W., & Page, R. (2014). BdcA, a protein important for Escherichia coli biofilm dispersal, is a short-chain dehydrogenase/reductase that binds specifically to NADPH. PloS one, 9(9), e105751.More infoThe Escherichia coli protein BdcA (previously referred to as YjgI) plays a key role in the dispersal of cells from bacterial biofilms, and its constitutive activation provides an attractive therapeutic target for dismantling these communities. In order to investigate the function of BdcA at a molecular level, we integrated structural and functional studies. Our 2.05 Å structure of BdcA shows that it is a member of the NAD(P)(H)-dependent short-chain dehydrogenase/reductase (SDR) superfamily. Structural comparisons with other members of the SDR family suggested that BdcA binds NADP(H). This was demonstrated experimentally using thermal denaturation studies, which showed that BcdA binds specifically to NADPH. Subsequent ITC experiments further confirmed this result and reported a Kd of 25.9 µM. Thus, BdcA represents the newest member of the limited number of oxidoreductases shown to be involved in quorum sensing and biofilm dispersal.
- Lord, D. M., Uzgoren Baran, A., Soo, V. W., Wood, T. K., Peti, W., & Page, R. (2014). McbR/YncC: implications for the mechanism of ligand and DNA binding by a bacterial GntR transcriptional regulator involved in biofilm formation. Biochemistry, 53(46), 7223-31.More infoMqsR-controlled colanic acid and biofilm regulator (McbR, also known as YncC) is the protein product of a highly induced gene in early Escherichia coli biofilm development and has been regarded as an attractive target for blocking biofilm formation. This protein acts as a repressor for genes involved in exopolysaccharide production and an activator for genes involved in stress response. To better understand the role of McbR in governing the switch from exponential growth to the biofilm state, we determined the crystal structure of McbR to 2.1 Å. The structure reveals McbR to be a member of the FadR C-terminal domain (FCD) family of the GntR superfamily of transcriptional regulators (this family was named after the first identified member, GntR, a transcriptional repressor of the gluconate operon of Bacillus subtilis). Previous to this study, only six of the predicted 2800 members of this family had been structurally characterized. Here, we identify the residues that constitute the McbR effector and DNA binding sites. In addition, comparison of McbR with other members of the FCD domain family shows that this family of proteins adopts highly distinct oligomerization interfaces, which has implications for DNA binding and regulation.
- Brown, B. L., Lord, D. M., Grigoriu, S., Peti, W., & Page, R. (2013). The Escherichia coli Toxin MqsR Destabilizes the Transcriptional Repression Complex Formed between the Antitoxin MqsA and the mqsRA Operon Promoter. JOURNAL OF BIOLOGICAL CHEMISTRY, 288(2), 1286-1294.
- Davis, J. R., Brown, B. L., Page, R., & Sello, J. K. (2013). Study of PcaV from Streptomyces coelicolor yields new insights into ligand-responsive MarR family transcription factors. Nucleic acids research, 41(6), 3888-900.More infoMarR family proteins constitute a group of >12 000 transcriptional regulators encoded in bacterial and archaeal genomes that control gene expression in metabolism, stress responses, virulence and multi-drug resistance. There is much interest in defining the molecular mechanism by which ligand binding attenuates the DNA-binding activities of these proteins. Here, we describe how PcaV, a MarR family regulator in Streptomyces coelicolor, controls transcription of genes encoding β-ketoadipate pathway enzymes through its interaction with the pathway substrate, protocatechuate. This transcriptional repressor is the only MarR protein known to regulate this essential pathway for aromatic catabolism. In in vitro assays, protocatechuate and other phenolic compounds disrupt the PcaV-DNA complex. We show that PcaV binds protocatechuate in a 1:1 stoichiometry with the highest affinity of any MarR family member. Moreover, we report structures of PcaV in its apo form and in complex with protocatechuate. We identify an arginine residue that is critical for ligand coordination and demonstrate that it is also required for binding DNA. We propose that interaction of ligand with this arginine residue dictates conformational changes that modulate DNA binding. Our results provide new insights into the molecular mechanism by which ligands attenuate DNA binding in this large family of transcription factors.
- Francis, D. M., Kumar, G. S., Koveal, D., Tortajada, A., Page, R., & Peti, W. (2013). The differential regulation of p38α by the neuronal kinase interaction motif protein tyrosine phosphatases, a detailed molecular study. Structure (London, England : 1993), 21(9), 1612-23.More infoThe MAP kinase p38α is essential for neuronal signaling. To better understand the molecular regulation of p38α we used atomistic and molecular techniques to determine the structural basis of p38α regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38α differently and their distinct molecular interactions explain their different catalytic activities. Although the interaction of PTPSL with p38α is similar to that of the previously described p38α:HePTP (PTPN7) complex, STEP binds and regulates p38α in an unexpected manner. Using NMR and small-angle X-ray scattering data, we generated a model of the p38α:STEP complex and define molecular differences between its resting and active states. Together, these results provide insights into molecular regulation of p38α by key regulatory proteins.
- Grigoriu, S., Bond, R., Cossio, P., Chen, J. A., Ly, N., Hummer, G., Page, R., Cyert, M. S., & Peti, W. (2013). The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin. PLOS BIOLOGY, 11(2).
- Koveal, D., Clarkson, M. W., Wood, T. K., Page, R., & Peti, W. (2013). Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas aeruginosa. JOURNAL OF MOLECULAR BIOLOGY, 425(12), 2219-2231.
- Koveal, D., Jayasundera, T. B., Wood, T. K., Peti, W., & Page, R. (2013). Backbone and sidechain H-1, N-15 and C-13 assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa. BIOMOLECULAR NMR ASSIGNMENTS, 7(1), 57-59.
- Kumar, G. S., Zettl, H., Page, R., & Peti, W. (2013). Structural basis for the regulation of the mitogen-activated protein (MAP) kinase p38α by the dual specificity phosphatase 16 MAP kinase binding domain in solution. The Journal of biological chemistry, 288(39), 28347-56.More infoMitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.
- Minnebo, N., Goernemann, J., O'Connell, N., Van, D. N., Derua, R., Vermunt, M. W., Page, R., Beullens, M., Peti, W., Van, E. A., & Bollen, M. (2013). NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes. NUCLEIC ACIDS RESEARCH, 41(2), 842-854.
- Peti, W., & Page, R. (2013). ENZYME MECHANISMS What's up 'Doc'?. NATURE CHEMICAL BIOLOGY, 9(12), 755-756.
- Peti, W., & Page, R. (2013). Molecular basis of MAP kinase regulation. PROTEIN SCIENCE, 22(12), 1698-1710.
- Peti, W., Nairn, A. C., & Page, R. (2013). Structural basis for protein phosphatase 1 regulation and specificity. FEBS JOURNAL, 280(2), 596-611.
- Wang, X., Lord, D. M., Hong, S. H., Peti, W., Benedik, M. J., Page, R., & Wood, T. K. (2013). Type II toxin/antitoxin MqsR/MqsA controls type V toxin/antitoxin GhoT/GhoS. Environmental microbiology, 15(6), 1734-44.More infoToxin endoribonucleases of toxin/antitoxin (TA) systems regulate protein production by selectively degrading mRNAs but have never been shown to control other TA systems. Here we demonstrate that toxin MqsR of the MqsR/MqsA system enriches toxin ghoT mRNA in vivo and in vitro, since this transcript lacks the primary MqsR cleavage site 5'-GCU. GhoT is a membrane toxin that causes the ghost cell phenotype, and is part of a type V TA system with antitoxin GhoS that cleaves specifically ghoT mRNA. Introduction of MqsR primary 5'-GCU cleavage sites into ghoT mRNA reduces ghost cell production and cell death likely due to increased degradation of the altered ghoT mRNA by MqsR. GhoT also prevents cell elongation upon the addition of low levels of ampicillin. Therefore, during stress, antitoxin GhoS mRNA is degraded by toxin MqsR allowing ghoT mRNA translation to yield another free toxin that forms ghost cells and increases persistence. Hence, we show that GhoT/GhoS is the first TA system regulated by another TA system.
- Choy, M. S., Page, R., & Peti, W. (2012). Regulation of protein phosphatase 1 by intrinsically disordered proteins. BIOCHEMICAL SOCIETY TRANSACTIONS, 40, 969-974.
- Eibl, C., Grigoriu, S., Hessenberger, M., Wenger, J., Puehringer, S., Pinheiro, A. S., Wagner, R. N., Proell, M., Reed, J. C., Page, R., Diederichs, K., & Peti, W. (2012). Structural and Functional Analysis of the NLRP4 Pyrin Domain. BIOCHEMISTRY, 51(37), 7330-7341.
- Koveal, D., Schuh-Nuhfer, N., Ritt, D., Page, R., Morrison, D. K., & Peti, W. (2012). A CC-SAM, for Coiled Coil-Sterile alpha Motif, Domain Targets the Scaffold KSR-1 to Specific Sites in the Plasma Membrane. SCIENCE SIGNALING, 5(255).
- O'Connell, N., Nichols, S. R., Heroes, E., Beullens, M., Bollen, M., Peti, W., & Page, R. (2012). The Molecular Basis for Substrate Specificity of the Nuclear NIPP1:PP1 Holoenzyme. STRUCTURE, 20(10), 1746-1756.
- Page, R., & Peti, W. (2012). Structural biology of MAPK (p38/ERK) regulation by phosphatases and scaffolding proteins. FASEB JOURNAL, 26.
- Peti, W., Nairn, A. C., & Page, R. (2012). Folding of Intrinsically Disordered Protein Phosphatase 1 Regulatory Proteins. Current physical chemistry, 2(1), 107-114.More infoIntrinsically disordered but biologically active proteins, commonly referred to as IDPs, are readily identified in many biological systems and play critical roles in multiple protein regulatory processes. While disordered in their unbound states, IDPs often, but not always, fold upon binding with their protein interaction partners. Here, we discuss how a class of IDPs directs the targeting, specificity and activity of Protein Phosphatase 1 (PP1). PP1 is major ser/thr phosphatase that plays a critical role in a broad range of biological processes, from muscle contraction to memory formation. In the cell, PP1 is regulated through its interaction with more than 200 regulatory proteins, the majority of which are IDPs. Critically, these PP1:regulatory protein holoenzyme complexes confer specificity to PP1 and are thus the functional forms of the PP1 enzyme in vivo. Furthermore, we discuss the distinct modes of interaction utilized by IDPs to complex with their protein binding partners. We subsequently show, by integrating multiple biophysical tools, that the majority of IDPs that regulate PP1, prefer a conformational selection model.
- Piserchio, A., Francis, D. M., Koveal, D., Dalby, K. N., Page, R., Peti, W., & Ghose, R. (2012). Docking Interactions of Hematopoietic Tyrosine Phosphatase with MAP Kinases ERK2 and p38 alpha. BIOCHEMISTRY, 51(41), 8047-8049.
- Piserchio, A., Francis, D. M., Koveal, D., Dalby, K. N., Page, R., Peti, W., & Ghose, R. (2012). Docking interactions of hematopoietic tyrosine phosphatase with MAP kinases ERK2 and p38α. Biochemistry, 51(41), 8047-9.More infoHematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both extracellular signal-regulated kinase (ERK) and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2-HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif also plays a key role in p38α-HePTP interactions. D-Motif-DRS interactions for the two kinases, while similar overall, do show some specific differences.
- Wang, X., Lord, D. M., Cheng, H., Osbourne, D. O., Hong, S. H., Sanchez-Torres, V., Quiroga, C., Zheng, K., Herrmann, T., Peti, W., Benedik, M. J., Page, R., & Wood, T. K. (2012). A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. NATURE CHEMICAL BIOLOGY, 8(10), 855-861.
- Brown, B. L., Wood, T. K., Peti, W., & Page, R. (2011). Structure of the Escherichia coli Antitoxin MqsA (YgiT/b3021) Bound to Its Gene Promoter Reveals Extensive Domain Rearrangements and the Specificity of Transcriptional Regulation. JOURNAL OF BIOLOGICAL CHEMISTRY, 286(3), 2285-2296.
- Brown, B. L., Wood, T. K., Peti, W., & Page, R. (2011). Structure of the Escherichia coli antitoxin MqsA (YgiT/b3021) bound to its gene promoter reveals extensive domain rearrangements and the specificity of transcriptional regulation. The Journal of biological chemistry, 286(3), 2285-96.More infoBacterial cultures, especially biofilms, produce a small number of persister cells, a genetically identical subpopulation of wild type cells that are metabolically dormant, exhibit multidrug tolerance, and are highly enriched in bacterial toxins. The gene most highly up-regulated in Escherichia coli persisters is mqsR, a ribonuclease toxin that, along with mqsA, forms a novel toxin·antitoxin (TA) system. Like all known TA systems, both the MqsR·MqsA complex and MqsA alone regulate their own transcription. Despite the importance of TA systems in persistence and biofilms, very little is known about how TA modules, and antitoxins in particular, bind and recognize DNA at a molecular level. Here, we report the crystal structure of MqsA bound to a 26-bp fragment from the mqsRA promoter. We show that MqsA binds DNA predominantly via its C-terminal helix-turn-helix domain, with direct binding of recognition helix residues Asn(97) and Arg(101) to the DNA major groove. Unexpectedly, the structure also revealed that the MqsA N-terminal domain interacts with the DNA phosphate backbone. This results in a more than 105° rotation of the N-terminal domains between the free and complexed states, an unprecedented rearrangement for an antitoxin. The structure also shows that MqsA bends the DNA by more than 55° in order to achieve symmetrical binding. Finally, using a combination of biochemical and NMR studies, we show that the DNA sequence specificity of MqsA is mediated by direct readout.
- Buckle, A. M., Bate, M. A., Androulakis, S., Cinquanta, M., Basquin, J., Bonneau, F., Chatterjee, D. K., Cittaro, D., Gräslund, S., Gruszka, A., Page, R., Suppmann, S., Wheeler, J. X., Agostini, D., Taussig, M., Taylor, C. F., Bottomley, S. P., Villaverde, A., & de Marco, A. (2011). Recombinant protein quality evaluation: proposal for a minimal information standard. Standards in genomic sciences, 5(2), 195-7.
- Dancheck, B., Ragusa, M. J., Allaire, M., Nairn, A. C., Page, R., & Peti, W. (2011). Molecular Investigations of the Structure and Function of the Protein Phosphatase 1-Spinophilin-Inhibitor 2 Heterotrimeric Complex. BIOCHEMISTRY, 50(7), 1238-1246.
- Dancheck, B., Ragusa, M. J., Allaire, M., Nairn, A. C., Page, R., & Peti, W. (2011). Molecular investigations of the structure and function of the protein phosphatase 1-spinophilin-inhibitor 2 heterotrimeric complex. Biochemistry, 50(7), 1238-46.More infoRegulation of the major Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry, and small-angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and inhibitor 2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1-spinophilin-I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1-spinophilin and PP1-I-2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1-I-2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multifunctional PP1 holoenzymes.
- Francis, D. M., Rozycki, B., Koveal, D., Hummer, G., Page, R., & Peti, W. (2011). Structural basis of p38 alpha regulation by hematopoietic tyrosine phosphatase. NATURE CHEMICAL BIOLOGY, 7(12), 916-924.
- Francis, D. M., Rozycki, B., Tortajada, A., Hummer, G., Peti, W., & Page, R. (2011). Resting and Active States of the ERK2:HePTP Complex. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 133(43), 17138-17141.
- Koveal, D., Pinheiro, A. S., Peti, W., & Page, R. (2011). Backbone and side chain H-1, N-15 and C-13 assignments of the KSR1 CA1 domain. BIOMOLECULAR NMR ASSIGNMENTS, 5(1), 39-41.
- Ragusa, M. J., Allaire, M., Nairn, A. C., Page, R., & Peti, W. (2011). Flexibility in the PP1: spinophilin holoenzyme. FEBS LETTERS, 585(1), 36-40.
- Wang, X., Kim, Y., Hong, S. H., Ma, Q., Brown, B. L., Pu, M., Tarone, A. M., Benedik, M. J., Peti, W., Page, R., & Wood, T. K. (2011). Antitoxin MqsA helps mediate the bacterial general stress response. NATURE CHEMICAL BIOLOGY, 7(6), 359-366.
- Brown, B. L., & Page, R. (2010). Preliminary crystallographic analysis of the Escherichia coli antitoxin MqsA (YgiT/b3021) in complex with mqsRA promoter DNA. Acta crystallographica. Section F, Structural biology and crystallization communications, 66(Pt 9), 1060-3.More infoThe Escherichia coli proteins MqsR and MqsA comprise a novel toxin-antitoxin (TA) system. MqsA, the antitoxin, defines a new family of antitoxins because unlike other antitoxins MqsA is structured throughout its entire sequence, binds zinc and coordinates DNA via its C-terminal and not its N-terminal domain. In order to understand how bacterial antitoxins, and MqsA in particular, regulate transcription, the MqsA protein was cocrystallized with a 26-mer duplex DNA corresponding to the palindromic region of the mqsRA promoter. The merohedrally twinned crystal belonged to space group P4(1), with unit-cell parameters a=60.99, b=60.99, c=148.60 A. A complete data set was collected to a resolution of 2.1 A. The solvent content of the crystal was consistent with the presence of two MqsA molecules bound to the duplex DNA in the asymmetric unit.
- Comeau, A. B., Critton, D. A., Page, R., & Seto, C. T. (2010). A focused library of protein tyrosine phosphatase inhibitors. Journal of medicinal chemistry, 53(18), 6768-72.More infoProtein tyrosine phosphatases such as PTP1B and YopH are potential targets for the development of therapeutic agents against a variety of pathological conditions including diabetes, obesity, and infection by the bacterium Yersinia pestis. A focused library of bidentate α-ketoacid-based inhibitors has been screened against several tyrosine phosphatases. Compound 2a has IC(50) values of 43 and 220 nM against YopH and PTP1B, respectively, and shows a 30-fold selectivity for PTP1B over the closely related phosphatase TCPTP.
- Kim, Y., Wang, X., Zhang, X., Grigoriu, S., Page, R., Peti, W., & Wood, T. K. (2010). Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD. ENVIRONMENTAL MICROBIOLOGY, 12(5), 1105-1121.
- Pinheiro, A. S., Proell, M., Eibl, C., Page, R., Schwarzenbacher, R., & Peti, W. (2010). Three-dimensional Structure of the NLRP7 Pyrin Domain INSIGHT INTO PYRIN-PYRIN-MEDIATED EFFECTOR DOMAIN SIGNALING IN INNATE IMMUNITY. JOURNAL OF BIOLOGICAL CHEMISTRY, 285(35), 27402-27410.
- Ragusa, M. J., Dancheck, B., Critton, D. A., Nairn, A. C., Page, R., & Peti, W. (2010). Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites. NATURE STRUCTURAL & MOLECULAR BIOLOGY, 17(4), 459-U100.
- Ragusa, M. J., Dancheck, B., Critton, D. A., Nairn, A. C., Page, R., & Peti, W. (2010). Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites. Nature structural & molecular biology, 17(4), 459-64.More infoThe serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with >or=200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1's specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form, and it binds PP1 through a folding-upon-binding mechanism in an elongated fashion, blocking one of PP1's three putative substrate binding sites without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1's activity toward a model substrate in vitro without affecting its ability to dephosphorylate its neuronal substrate, glutamate receptor 1 (GluR1). Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity.
- Brown, B. L., Grigoriu, S., Kim, Y., Arruda, J. M., Davenport, A., Wood, T. K., Peti, W., & Page, R. (2009). Three Dimensional Structure of the MqsR: MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties. PLOS PATHOGENS, 5(12).
- Brown, B. L., Grigoriu, S., Kim, Y., Arruda, J. M., Davenport, A., Wood, T. K., Peti, W., & Page, R. (2009). Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties. PLoS pathogens, 5(12), e1000706.More infoOne mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.
- Kelker, M. S., Page, R., & Peti, W. (2009). Crystal structures of protein phosphatase-1 bound to nodularin-R and tautomycin: a novel scaffold for structure-based drug design of serine/threonine phosphatase inhibitors. Journal of molecular biology, 385(1), 11-21.More infoProtein phosphatase 1 occurs in all tissues and regulates many pathways, ranging from cell-cycle progression to carbohydrate metabolism. Many naturally occurring, molecular toxins modulate PP1 activity, though the exact mechanism of this differential regulation is not understood. A detailed elucidation of these interactions is crucial for understanding the cellular basis of phosphatase function and signaling pathways but, more importantly, they can serve as the basis for highly specific therapeutics, e.g. against cancer. We report the crystal structures of PP1 in complex with nodularin-R at 1.63 A and tautomycin at 1.70 A resolution. The PP1:nodularin-R complex was used to demonstrate the utility of our improved PP1 production technique, which produces highly active, soluble PP1. Tautomycin is one of the few toxins that reportedly preferentially binds PP1>PP2A. Therefore, the PP1:tautomycin structure is the first complex structure with a toxin with preferred PP1 specificity. Furthermore, since tautomycin is a linear non-peptide-based toxin, our reported structure will aid the design of lead compounds for novel PP1-specific pharmaceuticals.
- Brown, B. L., Hadley, M., & Page, R. (2008). Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain. Protein expression and purification, 62(1), 9-14.More infoSpine-associated RapGAP (SPAR) is a 1783 residue, multidomain scaffolding protein which is a component of the NMDA receptor/PSD-95 complex in the post-synaptic density (PSD) of dendritic spines. Using a parallel expression screening approach, we identified a strategy to solubly express the SPAR PDZ domain in Escherichia coli. We show that maltose binding protein is required for the production of solubly expressed protein. We also show that small changes in construct length (2-5 residues) result in differential susceptibilities of the expressed proteins to proteolytic digestion, required for the expression tag removal. This has allowed us to identify a large-scale E. coli expression and purification protocol that results in the production of mg quantities of the SPAR PDZ domain. This is the first time that any of the multiple SPAR functional domains have been expressed in E. coli in quantities suitable for biophysical and biochemical studies, allowing us to investigate the role of the PDZ domain in SPAR function within the PSD.
- Critton, D., Tortajada, A., Stetson, G., Peti, W., & Page, R. (2008). Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase. BIOCHEMISTRY, 47(50), 13336-13345.
- Dugan, A. S., Maginnis, M. S., Jordan, J. A., Gasparovic, M. L., Manley, K., Page, R., Williams, G., Porter, E., O'Hara, B. A., & Atwood, W. J. (2008). Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells. The Journal of biological chemistry, 283(45), 31125-32.More infoBK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that alpha-defensin human neutrophil protein 1 (HNP1) and human alpha-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.
- Page, R. (2008). Strategies for improving crystallization success rates. Methods in molecular biology (Clifton, N.J.), 426, 345-62.More infoThe production of crystals suitable for high-resolution structure determination is still one of the major bottlenecks in the structure determination process. This is especially true in structural genomics (SG) consortia, where the implementation of protein-specific purification and optimization strategies is not readily implemented into the structure determination workflow. This chapter describes four strategies that have been implemented by a number of SG groups to increase the number of protein targets that resulted in atomic resolution structures: (1) orthologue screening; (2) the use of 1D (1)H NMR spectroscopy to screen for the folded state of a protein prior to crystallization; (3) deletion constructs generation, in which regions of the target protein predicted to be disordered are omitted from the construct, to maximize the likelihood of crystal formation; and (4) crystallization optimum solubility screening to identify more suitable buffers for a given protein. The implementation of these strategies can lead to a substantial increase in the number of protein structures solved. Finally, because these strategies do not require the implementation of expensive robotics, they are highly applicable not only for the SG community but also for academic laboratories.
- Lee, J., Page, R., Garcia-Contreras, R., Palermino, J., Zhang, X., Doshi, O., Wood, T. K., & Peti, W. (2007). Structure and function of the Escherichia coli protein YmgB: A protein critical for biofilm formation and acid-resistance. JOURNAL OF MOLECULAR BIOLOGY, 373(1), 11-26.
- Peti, W., & Page, R. (2007). Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. PROTEIN EXPRESSION AND PURIFICATION, 51(1), 1-10.
- Kosloff, M., Han, G. W., Krishna, S. S., Schwarzenbacher, R., Fasnacht, M., Elsliger, M. A., Abdubek, P., Agarwalla, S., Ambing, E., Astakhova, T., Axelrod, H. L., Canaves, J. M., Carlton, D., Chiu, H. J., Clayton, T., DiDonato, M., Duan, L., Feuerhelm, J., Grittini, C., , Grzechnik, S. K., et al. (2006). Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution. Proteins, 65(3), 527-37.More infoGlutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.
- Arndt, J. W., Schwarzenbacher, R., Page, R., Abdubek, P., Ambing, E., Biorac, T., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Godzik, A., Grittini, C., Grzechnik, S. K., Hale, J., Hampton, E., Han, G. W., Haugen, J., , Hornsby, M., et al. (2005). Crystal structure of an alpha/beta serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 A resolution. Proteins, 58(3), 755-8.
- Collins, B., Stevens, R. C., & Page, R. (2005). Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Acta crystallographica. Section F, Structural biology and crystallization communications, 61(Pt 12), 1035-8.More infoAn optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ;optimum-solubility' buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 A) structure determination.
- Han, G. W., Schwarzenbacher, R., Page, R., Jaroszewski, L., Abdubek, P., Ambing, E., Biorac, T., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Godzik, A., Grittini, C., Grzechnik, S. K., Hale, J., Hampton, E., Haugen, J., , Hornsby, M., et al. (2005). Crystal structure of an alanine-glyoxylate aminotransferase from Anabaena sp. at 1.70 A resolution reveals a noncovalently linked PLP cofactor. Proteins, 58(4), 971-5.
- Levin, I., Miller, M. D., Schwarzenbacher, R., McMullan, D., Abdubek, P., Ambing, E., Biorac, T., Cambell, J., Canaves, J. M., Chiu, H. J., Deacon, A. M., DiDonato, M., Elsliger, M. A., Godzik, A., Grittini, C., Grzechnik, S. K., Hale, J., Hampton, E., Han, G. W., , Haugen, J., et al. (2005). Crystal structure of an indigoidine synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 A resolution reveals a new fold. Proteins, 59(4), 864-8.
- Mustelin, T., Tautz, L., & Page, R. (2005). Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site. Journal of molecular biology, 354(1), 150-63.More infoHematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.
- Page, R., Deacon, A. M., Lesley, S. A., & Stevens, R. C. (2005). Shotgun crystallization strategy for structural genomics II: crystallization conditions that produce high resolution structures for T. maritima proteins. Journal of structural and functional genomics, 6(2-3), 209-17.More infoCurrently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664-11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally, 99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373-389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins, confirming its success for rapid identification of proteins with a natural propensity to crystallize.
- Page, R., Peti, W., Wilson, I. A., Stevens, R. C., & Wüthrich, K. (2005). NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline. Proceedings of the National Academy of Sciences of the United States of America, 102(6), 1901-5.More infoIn the Joint Center for Structural Genomics, one-dimensional (1D) 1H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D 1H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1D 1H NMR spectra, the proteins are classified into four groups, "A" to "D." A-type proteins are candidates for structure determination by NMR or crystallography; "B"-type are earmarked for crystallography; "C" indicates folded globular proteins with broadened line shapes; and "D" are nonglobular, "unfolded" polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D 1H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C- and D-type proteins.
- Peti, W., Johnson, M. A., Herrmann, T., Neuman, B. W., Buchmeier, M. J., Nelson, M., Joseph, J., Page, R., Stevens, R. C., Kuhn, P., & Wüthrich, K. (2005). Structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsP7. Journal of virology, 79(20), 12905-13.More infoHere, we report the three-dimensional structure of severe acute respiratory syndrome coronavirus (SARS-CoV) nsP7, a component of the SARS-CoV replicase polyprotein. The coronavirus replicase carries out regulatory tasks involved in the maintenance, transcription, and replication of the coronavirus genome. nsP7 was found to assume a compact architecture in solution, which is comprised primarily of helical secondary structures. Three helices (alpha2 to alpha4) form a flat up-down-up antiparallel alpha-helix sheet. The N-terminal segment of residues 1 to 22, containing two turns of alpha-helix and one turn of 3(10)-helix, is packed across the surface of alpha2 and alpha3 in the helix sheet, with the alpha-helical region oriented at a 60 degrees angle relative to alpha2 and alpha3. The surface charge distribution is pronouncedly asymmetrical, with the flat surface of the helical sheet showing a large negatively charged region adjacent to a large hydrophobic patch and the opposite side containing a positively charged groove that extends along the helix alpha1. Each of these three areas is thus implicated as a potential site for protein-protein interactions.
- Peti, W., Page, R., Moy, K., O'Neil-Johnson, M., Wilson, I. A., Stevens, R. C., & Wüthrich, K. (2005). Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR. Journal of structural and functional genomics, 6(4), 259-67.More infoIn structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D 1H NMR spectra or 2D [1H,15N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D 1H NMR and/or 2D [1H,15N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the "macro-scale" Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach.
- Xu, Q., Schwarzenbacher, R., McMullan, D., Abdubek, P., Ambing, E., Biorac, T., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Godzik, A., Grittini, C., Grzechnik, S. K., Hampton, E., Hornsby, M., Jaroszewski, L., Klock, H. E., , Koesema, E., et al. (2005). Crystal structure of a formiminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 A resolution reveals a new fold. Proteins, 58(4), 976-81.
- Bakolitsa, C., Schwarzenbacher, R., McMullan, D., Brinen, L. S., Canaves, J. M., Dai, X., Deacon, A. M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., , Kuhn, P., et al. (2004). Crystal structure of an orphan protein (TM0875) from Thermotoga maritima at 2.00-A resolution reveals a new fold. Proteins, 56(3), 607-10.
- Canaves, J. M., Page, R., Wilson, I. A., & Stevens, R. C. (2004). Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics. Journal of molecular biology, 344(4), 977-91.More infoCost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.
- Erlandsen, H., Canaves, J. M., Elsliger, M. A., von Delft, F., Brinen, L. S., Dai, X., Deacon, A. M., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., Kuhn, P., Lesley, S. A., McMullan, D., , McPhillips, T. M., et al. (2004). Crystal structure of an HEPN domain protein (TM0613) from Thermotoga maritima at 1.75 A resolution. Proteins, 54(4), 806-9.
- Heine, A., Canaves, J. M., von Delft, F., Brinen, L. S., Dai, X., Deacon, A. M., Elsliger, M. A., Eshaghi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Guda, C., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., , Kuhn, P., et al. (2004). Crystal structure of O-acetylserine sulfhydrylase (TM0665) from Thermotoga maritima at 1.8 A resolution. Proteins, 56(2), 387-91.
- Jaroszewski, L., Schwarzenbacher, R., von Delft, F., McMullan, D., Brinen, L. S., Canaves, J. M., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Hampton, E., Levin, I., Karlak, C., Klock, H. E., , Koesema, E., et al. (2004). Crystal structure of a novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 A resolution. Proteins, 56(3), 611-4.
- Levin, I., Schwarzenbacher, R., McMullan, D., Abdubek, P., Ambing, E., Biorac, T., Cambell, J., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Godzik, A., Grittini, C., Grzechnik, S. K., Hampton, E., Jaroszewski, L., Karlak, C., , Klock, H. E., et al. (2004). Crystal structure of a putative NADPH-dependent oxidoreductase (GI: 18204011) from mouse at 2.10 A resolution. Proteins, 56(3), 629-33.
- Levin, I., Schwarzenbacher, R., Page, R., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Campbell, J., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Hampton, E., , Jaroszewski, L., et al. (2004). Crystal structure of a PIN (PilT N-terminus) domain (AF0591) from Archaeoglobus fulgidus at 1.90 A resolution. Proteins, 56(2), 404-8.
- McMullan, D., Schwarzenbacher, R., Jaroszewski, L., von Delft, F., Klock, H. E., Vincent, J., Quijano, K., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshaghi, S., Floyd, R., Godzik, A., , Grittini, C., et al. (2004). Crystal structure of a novel Thermotoga maritima enzyme (TM1112) from the cupin family at 1.83 A resolution. Proteins, 56(3), 615-8.
- Miller, M. D., Schwarzenbacher, R., von Delft, F., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., , Hampton, E., et al. (2004). Crystal structure of a tandem cystathionine-beta-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 A resolution. Proteins, 57(1), 213-7.
- Page, R., & Stevens, R. C. (2004). Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens. Methods (San Diego, Calif.), 34(3), 373-89.More infoRecent efforts to collect and mine crystallization data from structural genomics (SG) consortia have led to the identification of minimal screens and novel screening strategies that can be used to streamline the crystallization process. Two groups, the Joint Center for Structural Genomics and the University of Toronto, carried out large-scale crystallization trials on different sets of bacterial targets (539, JCSG and 755, Toronto), using different sample processing and crystallization methods, and then analyzed their results to identify the smallest subset of conditions that would have crystallized the maximum number of protein targets. The JCSG Core Screen contains 67 conditions (from 480) while the Toronto Minimal Screen contains 6 (from 48). While the exact conditions included in the two screens do not overlap, the major precipitants of the conditions are similar and thus both screens can be used to determine if a protein has a natural propensity to crystallize. In addition, studies from other groups including the University of Queensland, the Mycobacterium tuberculosis SG group, the Southeast Collaboratory for SG, and the York Structural Biology Laboratory indicate that alternative crystallization strategies may be more successful at identifying initial crystallization conditions than typical sparse matrix screens. These minimal screens and alternative screening strategies are already being used to optimize the crystallization processes within large SG efforts. The differences between these results, however, demonstrate that additional studies which examine the influence of protein biophysical properties and sample preparation methods on crystal formation must also be carried out before more robust screens can be identified.
- Page, R., Moy, K., Sims, E. C., Velasquez, J., McManus, B., Grittini, C., Clayton, T. L., & Stevens, R. C. (2004). Scalable high-throughput micro-expression device for recombinant proteins. BioTechniques, 37(3), 364, 366, 368 passim.
- Page, R., Nelson, M. S., von Delft, F., Elsliger, M. A., Canaves, J. M., Brinen, L. S., Dai, X., Deacon, A. M., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., Kuhn, P., Lesley, S. A., , McMullan, D., et al. (2004). Crystal structure of gamma-glutamyl phosphate reductase (TM0293) from Thermotoga maritima at 2.0 A resolution. Proteins, 54(1), 157-61.
- Santelli, E., Schwarzenbacher, R., McMullan, D., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Dai, X., Deacon, A. M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., , Kovarik, J. S., et al. (2004). Crystal structure of a glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima (TM1621) at 1.60 A resolution. Proteins, 56(1), 167-70.
- Schwarzenbacher, R., Deacon, A. M., Jaroszewski, L., Brinen, L. S., Canaves, J. M., Dai, X., Elsliger, M. A., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., Kuhn, P., Lesley, S. A., McMullan, D., McPhillips, T. M., , Miller, M. D., et al. (2004). Crystal structure of a putative glutamine amido transferase (TM1158) from Thermotoga maritima at 1.7 A resolution. Proteins, 54(4), 801-5.
- Schwarzenbacher, R., Jaroszewski, L., von Delft, F., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., , Hampton, E., et al. (2004). Crystal structure of a type II quinolic acid phosphoribosyltransferase (TM1645) from Thermotoga maritima at 2.50 A resolution. Proteins, 55(3), 768-71.
- Schwarzenbacher, R., Jaroszewski, L., von Delft, F., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., , Hampton, E., et al. (2004). Crystal structure of an aspartate aminotransferase (TM1255) from Thermotoga maritima at 1.90 A resolution. Proteins, 55(3), 759-63.
- Schwarzenbacher, R., von Delft, F., Canaves, J. M., Brinen, L. S., Dai, X., Deacon, A. M., Elsliger, M. A., Eshaghi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Guda, C., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., Kovarik, J. S., Kreusch, A., , Kuhn, P., et al. (2004). Crystal structure of an iron-containing 1,3-propanediol dehydrogenase (TM0920) from Thermotoga maritima at 1.3 A resolution. Proteins, 54(1), 174-7.
- Schwarzenbacher, R., von Delft, F., Jaroszewski, L., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., , Hampton, E., et al. (2004). Crystal structure of a putative oxalate decarboxylase (TM1287) from Thermotoga maritima at 1.95 A resolution. Proteins, 56(2), 392-5.
- Spraggon, G., Schwarzenbacher, R., Kreusch, A., Lee, C. C., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Canaves, J. M., Cambell, J., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., , Grzechnik, S. K., et al. (2004). Crystal structure of an Udp-n-acetylmuramate-alanine ligase MurC (TM0231) from Thermotoga maritima at 2.3 A resolution. Proteins, 55(4), 1078-81.
- Spraggon, G., Schwarzenbacher, R., Kreusch, A., McMullan, D., Brinen, L. S., Canaves, J. M., Dai, X., Deacon, A. M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., Kovarik, J. S., , Kuhn, P., et al. (2004). Crystal structure of a methionine aminopeptidase (TM1478) from Thermotoga maritima at 1.9 A resolution. Proteins, 56(2), 396-400.
- Xu, Q., Schwarzenbacher, R., McMullan, D., von Delft, F., Brinen, L. S., Canaves, J. M., Dai, X., Deacon, A. M., Elsliger, M. A., Eshagi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., Jaroszewski, L., Karlak, C., Klock, H. E., Koesema, E., Kovarik, J. S., , Kreusch, A., et al. (2004). Crystal structure of a ribose-5-phosphate isomerase RpiB (TM1080) from Thermotoga maritima at 1.90 A resolution. Proteins, 56(1), 171-5.
- Xu, Q., Schwarzenbacher, R., Page, R., Sims, E., Abdubek, P., Ambing, E., Biorac, T., Brinen, L. S., Cambell, J., Canaves, J. M., Chiu, H. J., Dai, X., Deacon, A. M., DiDonato, M., Elsliger, M. A., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S. K., , Hampton, E., et al. (2004). Crystal structure of an allantoicase (YIR029W) from Saccharomyces cerevisiae at 2.4 A resolution. Proteins, 56(3), 619-24.
- Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., & Lesley, S. A. (2003). Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome. Acta crystallographica. Section D, Biological crystallography, 59(Pt 6), 1028-37.More infoAs the field of structural genomics continues to grow and new technologies are developed, novel strategies are needed to efficiently crystallize large numbers of protein targets, thus increasing output, not just throughput [Chayen & Saridakis (2002). Acta Cryst. D58, 921-927]. One strategy, developed for the high-throughput structure determination of the Thermotoga maritima proteome, is to quickly determine which proteins have a propensity for crystal formation followed by focused SeMet-incorporated protein crystallization attempts. This experimental effort has resulted in over 320 000 individual crystallization experiments. As such, it has provided one of the most extensive systematic data sets of commonly used crystallization conditions against a wide range of proteins to date. Analysis of this data shows that many of the original screening conditions are redundant, as all of the T. maritima proteins that crystallize readily could be identified using just 23% of the original conditions. It also shows that proteins that contain selenomethionine and are more extensively purified often crystallize in distinctly different conditions from those of their native less pure counterparts. Most importantly, it shows that the two-tiered strategy employed here is extremely successful for predicting which proteins will readily crystallize, as greater than 99% of the proteins identified as having a propensity to crystallize under non-optimal native conditions did so again as selenomethionine derivatives during the focused crystallization trials. This crystallization strategy can be adopted for both large-scale genomics programs and individual protein studies with multiple constructs and has the potential to significantly accelerate future crystallographic efforts.
- Nyman, T., Page, R., Schutt, C. E., Karlsson, R., & Lindberg, U. (2002). A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin. The Journal of biological chemistry, 277(18), 15828-33.More infoProfilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.
- Page, R., Lindberg, U., & Schutt, C. E. (1998). Domain motions in actin. Journal of molecular biology, 280(3), 463-74.More infoPrevious crystallographic investigations have shown that actin can undergo large conformational changes, even when complexed to the same actin binding protein. We have conducted a formal analysis of domain motions in actin, using the four available crystal structures, to classify the mechanism as either hinge or shear and to quantify the magnitude of these changes. We demonstrate that actin consists of two rigid cores, a semi-rigid domain and three conformationally variable extended loops. Confirming predictions about the nature of the domain rotation in actin based on its structural similarity to hexokinase, we show, using an algorithm previously used only to identify protein hinges, that residues at the interface between the two rigid cores undergo a shear between alternative conformations of actin. Rotations of less than 7 degrees in the torsion angles of five residues in the polypeptides that connect the rigid cores enable one actin conformation to be transformed into another. Because these torsion angle changes are small, the interface between the domains is maintained. In addition, we show that actin secondary structure elements, including those outside the rigid cores, are conformationally invariant among the four crystal structures, even when actin is complexed to different actin binding proteins. Finally, we demonstrate that the current F-actin models are inconsistent with the principles of actin conformational change identified here.
- Schutt, C. E., Kreatsoulas, C., Page, R., & Lindberg, U. (1997). Plugging into actin's architectonic socket. Nature structural biology, 4(3), 169-72.
- Page, R. (2017, April). Session Chair: Bacterial Persistence, Toxin-Antitoxin Systems and PrAMPs. In ASBMB Annual Meeting.
- Page, R. (2017, April). Session Chair: Microbiomes and Their Evolution During Infection and Disease. In ASBMB Annual Meeting.
- Page, R. (2017, April). Toxin-antitoxins systems and mRNA cleavage. In ASBMB Annual Meeting.
- Page, R. (2017, June 15). Toxin-antitoxins systems and mRNA cleavage. In 2017 Oklahoma University Annual Structural Biology Symposium.
- Page, R. (2016, April). Co-organizer: Protein Structure Dynamics and Function: Sailing the Protein Seas. In Protein Structure Dynamics and Function: Sailing the Protein Seas.
- Page, R. (2016, July). How RepoMan (and Ki-67) direct PP1γ to chromosomes at anaphase onset. In 2016 FASEB Meeting: Protein phosphatases.
- Page, R. (2016, November). Phosphatases and SLiMs. In 2016 V Latin American Protein Society Meeting.
- Page, R. (2015, April). Making ser/thr phosphatases (PP1, calcineurin) druggable: achieving specificity by targeting substrate and regulatory protein interaction sites. In 2015 ABBIX-NSLSII National Scientific Meeting.
- Page, R. (2015, April). Toxin-antitoxin systems and their role(s) in persistence. In 2015 ASBMB National Scientific Meeting.
- Page, R. (2018, Feb). Cracking the phosphatase code: Repoman and Ki67. Therapeutic Development Meeting Seminar Series. Tucson, Arizona: UA Cancer Center.
- Page, R. (2016, January). Cracking the Phosphatase Code. University of Arizona, invited seminar.
- Page, R. (2015, Fall). How PP1 holoenzymes drive mitotic exit. Brunel University of London Seminar Series.
- Page, R. (2015, November). How PP1 holoenzymes drive mitotic exit. University of Leuven Seminar Series.
- Page, R. (2015, October). MqsRA: the structural basis of biofilm formation and antibiotic resistance. Case Western Reserve University Seminar Series.
- Page, R. (2014, February). Using Structure to Unravel the Molecular Basis of Persistence & Antibiotic Resistance in Bacteria. University of Bayreuth Faculty Seminar.
- Page, R. (2014, June). Using structure to understand signaling in prokaryotic and eukaryotic systems. Copenhagen University Faculty Seminar.
- Page, R. (2014, March). Kinases and phosphatases: structural insights into specificity. URI Faculty Seminar.
- Page, R. (2014, March). Protein activation by PNUTS provides new insights into the Protein Phosphatase 1 Regulatory Code. Rutgers University Center for Integrative Proteomics Research.