- Professor, Chemistry and Biochemistry
- Professor, Chemistry and Biochemistry-Sci
- Professor, BIO5 Institute
1992 – 1998 Diploma Magister rer. nat, Chemistry, summa cum laude, University of Vienna, Austria
1/1997 – 12/1997: Diploma thesis
Laboratory of Dr.Dr. B.K. Keppler, Department of Chemistry, University of Vienna, Austria: Synthesis, Characterization and Investigation of the Hydrolysis of Tumor Inhibiting Ruthenium Complexes
4/1998 – 9/2001 Ph.D., Chemistry, summa cum laude, J.-W.-G. University Frankfurt, Germany
Laboratory of Dr. C. Griesinger, Department of Chemistry, J.-W.-G. University Frankfurt, Germany: New Methods for the Elucidation of NMR Projection Restraints: Structure and Dynamic of Native and Denatured Proteins
10/2001 – 8/2004 Research Associate, The Scripps Research Institute, USA
Laboratory of Dr. K. Wüthrich, Department of Molecular Biology, The Scripps Research Institute: Structural Proteomics using NMR Spectroscopy; Novel NMR Screening Techniques; Structure Determination of Proteomic Target Proteins
9/2004 – 6/2010 Assistant Professor of Medical Science, MPPB, Brown University, USA
7/2006 – 8/2010 MPP Graduate Program Director, Brown University, USA
7/2007 – 6/2010 Manning Assistant Professor, MPPB, Brown University, USA
7/2008 – 6/2010 Assistant Professor of Chemistry, Brown University, USA
7/2010 – 6/2015 Associate Professor (tenure) of Medical Science, Brown University, USA
7/2010 – 6/2015 Associate Professor (tenure) of Chemistry, Brown University, USA
7/2012 – 12/2016 Director Structural Biology Core Facility, Brown University, USA
11/2014 – present Affiliated Professor of Biology, University of Copenhagen, Denmark
7/2015 – 12/2016 Professor (tenure) of Medical Science, Brown University, USA
7/2015 – 12/2016 Professor (tenure) of Chemistry, Brown University, USA
1/2017 – present Professor (tenure) of Chemistry and Biochemistry, University of Arizona, USA
1/2017 – present Member Bio5 Institute, University of Arizona, USA
- Ph.D. Chemistry
- University of Frankfurt, Frankfurt, Germany
- New Methods for the Elucidation of NMR Projection Restraints: Structure and Dynamic of Native and Denatured Proteins
- M.S. Chemistry
- University of Vienna, Vienna, Austria
- Synthesis, Characterization and Investigation of the Hydrolysis of Tumor Inhibiting Ruthenium Complexes
- Professor, University of Arizona, Tucson, Arizona (2017 - Ongoing)
- Affiliated Professor of Biology, University of Copenhagen (2014 - Ongoing)
- Professor, Brown University, Providence, Rhode Island (2004 - 2016)
- Homer C. and Emily Davis Weed Chair
- University of Arizona, Spring 2017
- Pathway to Stop Diabetes
- American Diabetes Association, Spring 2014
undergraduate and graduate education
Signaling Enzymes, Structural Biology, Chemical Biology, NMR spectroscopy, Structure-function relationships, protein dynamics, allostery, novel routes for drug design
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- Choy, M. S., Li, Y., Machado, L. E., Kunze, M. B., 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), 644-658.e5.More infoProtein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function.
- 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 Munter, S., Bollen, M., Vagnarelli, P., Peti, W., & Page, R. (2016). The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism. eLife, 5.More infoKi-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.
- Page, R., & Peti, W. (2016). Toxin-antitoxin systems in bacterial growth arrest and persistence. Nature chemical biology, 12(4), 208-14.More infoBacterial persister cells constitute a subpopulation of genetically identical, metabolically slow-growing cells that are highly tolerant of antibiotics and other environmental stresses. Recent studies have demonstrated that gene loci known as toxin-antitoxin (TA) modules play a central role in the persister state. Under normal growth conditions, antitoxins potently inhibit the activities of the toxins. In contrast, under conditions of stress, the antitoxins are selectively degraded, freeing the toxins to inhibit essential cellular processes, such as DNA replication and protein translation. This inhibition results in rapid growth arrest. In this Review, we highlight recent discoveries of these multifaceted TA systems with a focus on the newly uncovered mechanisms, especially conditional cooperativity, that are used to regulate cell growth and persistence. We also discuss the potential for targeting TA systems for antimicrobial drug discovery.
- 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 πɸLxVP SLiM. Scientific reports, 6, 38920.More infoSer/thr phosphorylation is the primary reversible covalent modification of proteins in eukaryotes. As a consequence, it is the reciprocal actions of kinases and phosphatases that act as key molecular switches to fine tune cellular events. It has been well documented that ~400 human ser/thr kinases engage substrates via consensus phosphosite sequences. Strikingly, we know comparatively little about the mechanism by which ~40 human protein ser/thr phosphatases (PSPs) dephosphorylate ~15000 different substrates with high specificity. The identification of substrates of the essential PSP calcineurin (CN) has been exceptionally challenging and only a small fraction has been biochemically confirmed. It is now emerging that CN binds regulators and substrates via two short linear motifs (SLiMs), the well-studied PxIxIT SLiM and the LxVP SLiM, which remains controversial at the molecular level. Here we describe the crystal structure of CN in complex with its substrate NFATc1 and show that the LxVP SLiM is correctly defined as πɸLxVP. Bioinformatics studies using the πɸLxVP SLiM resulted in the identification of 567 potential CN substrates; a small subset was experimentally confirmed. This combined structural-bioinformatics approach provides a powerful method for dissecting the CN interaction network and for elucidating the role of CN in human health and disease.
- Wang, X., Bajaj, R., Bollen, M., Peti, W., & Page, R. (2016). Expanding the PP2A Interactome by Defining a B56-Specific SLiM. Structure (London, England : 1993), 24(12), 2174-2181.More infoSpecific interactions between proteins govern essential physiological processes including signaling. Many enzymes, especially the family of serine/threonine phosphatases (PSPs: PP1, PP2A, and PP2B/calcineurin/CN), recruit substrates and regulatory proteins by binding short linear motifs (SLiMs), short sequences found within intrinsically disordered regions that mediate specific protein-protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PSPs, especially PP1 and CN, essentially nothing is known about how SLiMs bind PP2A, a validated cancer drug target. Here we describe three structures of a PP2A-SLiM interaction (B56:pS-RepoMan, B56:pS-BubR1, and B56:pSpS-BubR1), show that this PP2A-specific SLiM is defined as LSPIxE, and then use these data to discover scores of likely PP2A regulators and substrates. Together, these data provide a powerful approach not only for dissecting PP2A interaction networks in cells but also for targeting PP2A diseases, such as cancer.
- 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 eIF2α Phosphatase. Cell reports, 11(12), 1885-91.More infoThe attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.
- Krishnan, N., Krishnan, K., Connors, C. R., Choy, M. S., Page, R., Peti, W., Van Aelst, L., Shea, S. D., & Tonks, N. K. (2015). PTP1B inhibition suggests a therapeutic strategy for Rett syndrome. The Journal of clinical investigation, 125(8), 3163-77.More infoThe X-linked neurological disorder Rett syndrome (RTT) presents with autistic features and is caused primarily by mutations in a transcriptional regulator, methyl CpG-binding protein 2 (MECP2). Current treatment options for RTT are limited to alleviating some neurological symptoms; hence, more effective therapeutic strategies are needed. We identified the protein tyrosine phosphatase PTP1B as a therapeutic candidate for treatment of RTT. We demonstrated that the PTPN1 gene, which encodes PTP1B, was a target of MECP2 and that disruption of MECP2 function was associated with increased levels of PTP1B in RTT models. Pharmacological inhibition of PTP1B ameliorated the effects of MECP2 disruption in mouse models of RTT, including improved survival in young male (Mecp2-/y) mice and improved behavior in female heterozygous (Mecp2-/+) mice. We demonstrated that PTP1B was a negative regulator of tyrosine phosphorylation of the tyrosine kinase TRKB, the receptor for brain-derived neurotrophic factor (BDNF). Therefore, the elevated PTP1B that accompanies disruption of MECP2 function in RTT represents a barrier to BDNF signaling. Inhibition of PTP1B led to increased tyrosine phosphorylation of TRKB in the brain, which would augment BDNF signaling. This study presents PTP1B as a mechanism-based therapeutic target for RTT, validating a unique strategy for treating the disease by modifying signal transduction pathways with small-molecule drugs.
- 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), 2781-5.More infoThe established dogma is that protein serine/threonine (PSPs) and tyrosine (PTPs) phosphatases are unattainable drug targets. This is because natural product inhibitors of PSP active sites are lethal, while the active sites of PTPs are exceptionally conserved and charged, making it nearly impossible to develop PTP inhibitors that are selective. However, due to a series of recent structural and functional studies, this view of phosphatases is about to undergo a radical change. Rather than target active sites, these studies have demonstrated that targeting PSP/PTP protein (substrate/regulatory) interaction sites, which are distal from the active sites, are highly viable and suitable drugs targets. This is especially true for calcineurin (CN), in which the blockbuster immunosuppressant drugs FK506 and cyclosporin A were recently demonstrated to bind and block one of the key CN substrate interaction sites, the LxVP site. Additional studies show that this approach-targeting substrate and/or regulatory protein interaction sites-also holds incredible promise for protein phosphatase 1 (PP1)-related diseases. Finally, domains outside PTP catalytic domains have also recently been demonstrated to directly alter PTP activity. Collectively, these novel insights offer new, transformative perspectives for the therapeutic targeting of PSPs by interfering with the binding of PIPs or substrates and PTPs by targeting allosteric sites outside their catalytic domains.
- 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), 4097-102.More infoThe serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.
- Krishnan, N., Koveal, D., Miller, D. H., Xue, B., Akshinthala, S. D., Kragelj, J., Jensen, M. R., Gauss, C. M., 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), 558-66.More infoPTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, noncatalytic segment of PTP1B. We present what is to our knowledge the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small-molecule inhibitor MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.
- 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.
- 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.
- Peti, W., & Page, R. (2013). Enzyme mechanisms: What's up 'Doc'?. Nature chemical biology, 9(12), 756-7.
- Peti, W., & Page, R. (2013). Molecular basis of MAP kinase regulation. Protein science : a publication of the Protein Society, 22(12), 1698-710.More infoMitogen-activated protein kinases (MAPKs; ERK1/2, p38, JNK, and ERK5) have evolved to transduce environmental and developmental signals (growth factors, stress) into adaptive and programmed responses (differentiation, inflammation, apoptosis). Almost 20 years ago, it was discovered that MAPKs contain a docking site in the C-terminal lobe that binds a conserved 13-16 amino acid sequence known as the D- or KIM-motif (kinase interaction motif). Recent crystal structures of MAPK:KIM-peptide complexes are leading to a precise understanding of how KIM sequences contribute to MAPK selectivity. In addition, new crystal and especially NMR studies are revealing how residues outside the canonical KIM motif interact with specific MAPKs and contribute further to MAPK selectivity and signaling pathway fidelity. In this review, we focus on these recent studies, with an emphasis on the use of NMR spectroscopy, isothermal titration calorimetry and small angle X-ray scattering to investigate these processes.