Loop Dynamics and Enzyme Catalysis in Protein Tyrosine Phosphatases

Protein tyrosine phosphatases (PTPs) play an important role in cellular signalling and have been implicated in human cancers, diabetes, and obesity. Despite shared catalytic mechanisms and transition states for the chemical steps of catalysis, catalytic rates within the PTP family vary over several orders of magnitude. These rate differences have been implied to arise from differing conformational dynamics of the closure of a protein loop, the WPD-loop, which carries a catalytically critical residue. The present work reports computational studies of the human protein tyrosine phosphatase 1B (PTP1B), and YopH from Yersinia pestis, for which NMR has demonstrated a link between both their respective rates of WPD-loop motion and catalysis rates, which differ by an order of magnitude. We have performed detailed structural analysis, both conventional and enhanced sampling simulations of their loop dynamics, as well as empirical valence bond simulations of the chemical step of catalysis. These analyses revealed the key residues and structural features responsible for these differences, as well as the residues and pathways that facilitate allosteric communication in these enzymes. Curiously, our wild-type YopH simulations also identify a catalytically incompetent hyper-open conformation of its WPD-loop, sampled as a rare event, previously only experimentally observed in YopH-based chimeras. The effect of differences within the WPD-loop and its neighbouring loops on the modulation of loop dynamics, as revealed in this work, may provide a facile means for the family of PTP enzymes to respond to environmental changes and regulate their catalytic activities.

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Loop Dynamics and Enzyme Catalysis in Protein Tyrosine Phosphatases

10.26434/chemrxiv.13217432.v2 ◽

2021 ◽

Author(s):

Rory Crean ◽

Michal Biler ◽

Marc van der Kamp ◽

Alvan C. Hengge ◽

Shina Caroline Lynn Kamerlin

Keyword(s):

Environmental Changes ◽

Tyrosine Phosphatase ◽

Conformational Dynamics ◽

Rare Event ◽

Protein Tyrosine Phosphatases ◽

Structural Features ◽

Tyrosine Phosphatases ◽

Protein Tyrosine ◽

Critical Residue ◽

Loop Dynamics

Protein tyrosine phosphatases (PTPs) play an important role in cellular signalling and have been implicated in human cancers, diabetes, and obesity. Despite shared catalytic mechanisms and transition states for the chemical steps of catalysis, catalytic rates within the PTP family vary over several orders of magnitude. These rate differences have been implied to arise from differing conformational dynamics of the closure of a protein loop, the WPD-loop, which carries a catalytically critical residue. The present work reports computational studies of the human protein tyrosine phosphatase 1B (PTP1B), and YopH from Yersinia pestis, for which NMR has demonstrated a link between both their respective rates of WPD-loop motion and catalysis rates, which differ by an order of magnitude. We have performed detailed structural analysis, both conventional and enhanced sampling simulations of their loop dynamics, as well as empirical valence bond simulations of the chemical step of catalysis. These analyses revealed the key residues and structural features responsible for these differences, as well as the residues and pathways that facilitate allosteric communication in these enzymes. Curiously, our wild-type YopH simulations also identify a catalytically incompetent hyper-open conformation of its WPD-loop, sampled as a rare event, previously only experimentally observed in YopH-based chimeras. The effect of differences within the WPD-loop and its neighbouring loops on the modulation of loop dynamics, as revealed in this work, may provide a facile means for the family of PTP enzymes to respond to environmental changes and regulate their catalytic activities.

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Insights into the Importance of WPD-Loop Sequence for Activity and Structure in Protein Tyrosine Phosphatases

10.33774/chemrxiv-2021-8f6mc ◽

2021 ◽

Author(s):

Ruidan Shen ◽

Rory M. Crean ◽

Keith J. Olsen ◽

Teisha Richan ◽

Tiago A. S. Brandão ◽

...

Keyword(s):

Structural Integrity ◽

Conformational Dynamics ◽

Chimeric Protein ◽

Protein Tyrosine Phosphatases ◽

Tyrosine Phosphatases ◽

Protein Tyrosine ◽

Loop Sequence ◽

Loop Dynamics ◽

Catalytic Loop ◽

Time Required

Protein tyrosine phosphatases (PTPs) possess a mobile, conserved catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. Also, work on Chimeras of YopH bearing parts of the WPD-loop sequence from PTP1B demonstrated unusual structural perturbations and reduced activity. In the present study, we have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics in catalysis. These chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, despite unaltered chemical mechanisms and transition states. The chimeric proteins’ WPD-loops differ significantly in their relative stability and rigidity. In particular, the open WPD-loops sample multiple metastable and interconverting conformations. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. These differences in loop dynamics affect both the pH dependency of catalysis and turnover rate. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

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Design and Biological Evaluation of Novel Imidazolyl Flavonoids as Potent and Selective Protein Tyrosine Phosphatase Inhibitors

Medicinal Chemistry ◽

10.2174/1573406415666190430125547 ◽

2020 ◽

Vol 16 (4) ◽

pp. 563-574 ◽

Cited By ~ 1

Author(s):

Rong Y. Han ◽

Yu Ge ◽

Ling Zhang ◽

Qing M. Wang

Keyword(s):

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Protein Tyrosine Phosphatases ◽

Structural Features ◽

Biological Evaluation ◽

Cell Protein ◽

Phosphatase Inhibitors ◽

Protein Tyrosine ◽

Therapeutic Development ◽

Chemical Studies

Background: Protein tyrosine phosphatases 1B are considered to be a desirable validated target for therapeutic development of type II diabetes and obesity. Methods: A new series of imidazolyl flavonoids as potential protein tyrosine phosphatase inhibitors were synthesized and evaluated. Results: Bioactive results indicated that some synthesized compounds exhibited potent protein phosphatase 1B (PTP1B) inhibitory activities at the micromolar range. Especially, compound 8b showed the best inhibitory activity (IC50=1.0 µM) with 15-fold selectivity for PTP1B over the closely related T-cell protein tyrosine phosphatase (TCPTP). Cell viability assays indicated that 8b is cell permeable with lower cytotoxicity. Molecular modeling and dynamics studies revealed the reason for selectivity of PTP1B over TCPTP. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity. Conclusion: Compound 8b should be a potential selective PTP1B inhibitor.

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Targeting Tyrosine Phosphatases by 3-Bromopyruvate Overcomes Hyperactivation of Platelets from Gastrointestinal Cancer Patients

Journal of Clinical Medicine ◽

10.3390/jcm8070936 ◽

2019 ◽

Vol 8 (7) ◽

pp. 936 ◽

Cited By ~ 6

Author(s):

Faria ◽

Andrade ◽

Reijm ◽

Spaander ◽

de Maat ◽

...

Keyword(s):

Tumor Cell ◽

Cancer Patients ◽

Gastrointestinal Cancer ◽

Molecular Mechanisms ◽

Tyrosine Phosphatase ◽

Protein Tyrosine Phosphatases ◽

Cell Aggregation ◽

Tyrosine Phosphatases ◽

Indirect Contact ◽

Protein Tyrosine

Venous thromboembolism (VTE) is one of the most common causes of cancer related mortality. It has been speculated that hypercoagulation in cancer patients is triggered by direct or indirect contact of platelets with tumor cells, however the underlying molecular mechanisms involved are currently unknown. Unraveling these mechanisms may provide potential avenues for preventing platelet-tumor cell aggregation. Here, we investigated the role of protein tyrosine phosphatases in the functionality of platelets in both healthy individuals and patients with gastrointestinal cancer, and determined their use as a target to inhibit platelet hyperactivity. This is the first study to demonstrate that platelet agonists selectively activate low molecular weight protein tyrosine phosphatase (LMWPTP) and PTP1B, resulting in activation of Src, a tyrosine kinase known to contribute to several platelet functions. Furthermore, we demonstrate that these phosphatases are a target for 3-bromopyruvate (3-BP), a lactic acid analog currently investigated for its use in the treatment of various metabolic tumors. Our data indicate that 3-BP reduces Src activity, platelet aggregation, expression of platelet activation makers and platelet-tumor cell interaction. Thus, in addition to its anti-carcinogenic effects, 3-BP may also be effective in preventing platelet-tumor cell aggregationin cancer patients and therefore may reduce cancer mortality by limiting VTE in patients.

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Multiple Functions of the Eya Phosphotyrosine Phosphatase

Molecular and Cellular Biology ◽

10.1128/mcb.00976-15 ◽

2015 ◽

Vol 36 (5) ◽

pp. 668-677 ◽

Cited By ~ 20

Author(s):

Ilaria Rebay

Keyword(s):

Developmental Regulation ◽

Tyrosine Phosphatase ◽

Protein Tyrosine Phosphatases ◽

Transcriptional Coactivator ◽

Tyrosine Phosphatases ◽

Phosphotyrosine Phosphatase ◽

Eyes Absent ◽

Multiple Functions ◽

Protein Tyrosine ◽

Cellular Events

Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling.

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Podocyte Protein, Nephrin, Is a Substrate of Protein Tyrosine Phosphatase 1B

Journal of Signal Transduction ◽

10.1155/2011/376543 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 21

Author(s):

Lamine Aoudjit ◽

Ruihua Jiang ◽

Tae Hoon Lee ◽

Laura A. New ◽

Nina Jones ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Tyrosine Phosphorylation ◽

Tyrosine Phosphatase ◽

Protein Tyrosine Phosphatases ◽

Protein Tyrosine Phosphatase 1B ◽

Tyrosine Phosphatases ◽

Puromycin Aminonucleoside ◽

Protein Tyrosine ◽

Aminonucleoside Nephrosis ◽

Puromycin Aminonucleoside Nephrosis

Glomerular podocytes are critical for the barrier function of the glomerulus in the kidney and their dysfunction causes protein leakage into the urine (proteinuria). Nephrin is a key podocyte protein, which regulates the actin cytoskeleton via tyrosine phosphorylation of its cytoplasmic domain. Here we report that two protein tyrosine phosphatases, PTP1B and PTP-PEST negatively regulate nephrin tyrosine phosphorylation. PTP1B directly binds to and dephosphorylates nephrin, while the action of PTP-PEST is indirect. The two phosphatases are also upregulated in the glomerulus in the rat model of puromycin aminonucleoside nephrosis. Both overexpression and inhibition of PTP1B deranged the actin cytoskeleton in cultured mouse podocytes. Thus, protein tyrosine phosphatases may affect podocyte function via regulating nephrin tyrosine phosphorylation.

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Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.836 ◽

1992 ◽

Vol 12 (2) ◽

pp. 836-846 ◽

Cited By ~ 229

Author(s):

T L Yi ◽

J L Cleveland ◽

J N Ihle

Keyword(s):

Tyrosine Phosphorylation ◽

Protein Tyrosine Phosphatase ◽

Human Gene ◽

Tyrosine Phosphatase ◽

Hematopoietic Cells ◽

Protein Tyrosine Phosphatases ◽

Hematopoietic Cell ◽

Protein Tyrosine Phosphorylation ◽

Tyrosine Phosphatases ◽

Protein Tyrosine

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.

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A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

10.26434/chemrxiv.13604504.v1 ◽

2021 ◽

Author(s):

Ruidan Shen ◽

Rory Crean ◽

Sean Johnson ◽

Shina Caroline Lynn Kamerlin ◽

Alvan C. Hengge

Keyword(s):

Conformational Equilibrium ◽

Protein Tyrosine Phosphatases ◽

Tyrosine Phosphatases ◽

Ph Profile ◽

Catalytic Residues ◽

Protein Tyrosine ◽

Closed Conformation ◽

Ph Dependency ◽

Loop Dynamics ◽

Rate Profile

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic pKa of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

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A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

10.26434/chemrxiv.13604504.v2 ◽

2021 ◽

Author(s):

Ruidan Shen ◽

Rory Crean ◽

Sean Johnson ◽

Shina Caroline Lynn Kamerlin ◽

Alvan C. Hengge

Keyword(s):

Conformational Equilibrium ◽

Protein Tyrosine Phosphatases ◽

Tyrosine Phosphatases ◽

Ph Profile ◽

Catalytic Residues ◽

Protein Tyrosine ◽

Closed Conformation ◽

Ph Dependency ◽

Loop Dynamics ◽

Rate Profile

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic pKa of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

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