Yersinia Protein Tyrosine Phosphatase Research Articles

On the recognition of Yersinia protein tyrosine phosphatase by carboxylic acid derivatives

Some members of Yersinia (Y), a genus of bacteria in the family Yersiniaceae, are pathogenic in humans, causing a range of health problems, from gastrointestinal syndromes to the plague. The Y protein tyrosine phosphatase (PTP) YopH is a crucial virulence determinant, considering the vital roles of PTPs in the intracellular signal transduction pathways and cell cycle control. The structural understanding of YopH as a cellular target in pathogenic conditions caused by Y infection is a prerequisite for designing potent and selective YopH inhibitors. Thus, by using molecular docking simulations, the open and closed conformations of the so-called ‘WPD loop’ (352-Gly-Asn-Trp-Pro-Asp-Gln-Thr-Ala-Val-Ser-361), located nearby the active site (403-Cys-Arg-Ala-Gly-Val-Gly-Arg-Thr-410) in YopH structure, are shown to be relevant for recognition by carboxylic acid derivatives, and the closed conformation is a more preferable receptor in terms of the quantitative correlation with experimental data. In both cases, aurintricarboxylic acid (ATA) has the greatest affinity to YopH. Consequently, a quantum mechanics/molecular mechanics (QM/MM) molecular model is derived to see into the extent of the ATA-induced open-closed conformational change. Active site residues and the WPD loop, as well as ATA are treated using SCC-DFTB-D (QM level), while the rest of the complex is treated using AMBER force field (MM level). The active/inactive functional behavior of YopH is explored by observing the interaction mode of ATA with the wild-type (wt)/Cys403Ser receptor and evaluating the competitive inhibition parameters. Implications of the present study for experimental research are discussed.

Computational Studies of Catalytic Loop Dynamics in Yersinia Protein Tyrosine Phosphatase Using Pathway Optimization Methods.

Yersinia Protein Tyrosine Phosphatase (YopH) is the most efficient enzyme among all known PTPases and relies on its catalytic loop movements for substrate binding and catalysis. Fluorescence, NMR, and UV resonance Raman (UVRR) techniques have been used to study the thermodynamic and dynamic properties of the loop motions. In this study, a computational approach based on the pathway refinement methods nudged elastic band (NEB) and harmonic Fourier beads (HFB) has been developed to provide structural interpretations for the experimentally observed kinetic processes. In this approach, the minimum potential energy pathways for the loop open/closure conformational changes were determined by NEB using a one-dimensional global coordinate. Two dimensional data analyses of the NEB results were performed as an efficient method to qualitatively evaluate the energetics of transitions along several specific physical coordinates. The free energy barriers for these transitions were then determined more precisely using the HFB method. Kinetic parameters were estimated from the energy barriers using transition state theory and compared against experimentally determined kinetic parameters. When the calculated energy barriers are calibrated by a simple "scaling factor", as have been done in our previous vibrational frequency calculations to explain the ligand frequency shift upon its binding to protein, it is possible to make structural interpretations of several observed enzyme dynamic rates. For example, the nanosecond kinetics observed by fluorescence anisotropy may be assigned to the translational motion of the catalytic loop and microsecond kinetics observed in fluorescence T-jump can be assigned to the loop backbone dihedral angle flipping. Furthermore, we can predict that a Trp354 conformational conversion associated with the loop movements would occur on the tens of nanoseconds time scale, to be verified by future UVRR T-jump studies.

1H, 15N, and 13C backbone resonance assignments for the yersinia protein tyrosine phosphatase YopH

YopH is a protein tyrosine phosphatase that functions as a required virulence factor in Yersinia. Here we report the backbone resonance assignments for a point mutant of the C-terminal catalytic domain of YopH.

Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.

Investigation of Catalytic Loop Structure, Dynamics, and Function Relationship ofYersiniaProtein Tyrosine Phosphatase by Temperature-Jump Relaxation Spectroscopy and X-ray Structural Determination

Yersinia protein tyrosine phosphatase (YopH) is the most efficient enzyme among all PTPases. YopH is hyperactive compared to human PTPases, interfering with mammalian cellular pathways to achieve the pathogenicity of Yersinia. Two properties related to the catalytic loop structure differences have been proposed to affect its dynamics and enzyme efficiency. One is the ability of the loop to form stabilizing interactions to bound ligand after loop closure, which has long been recognized. In addition, the loop flexibility/mobility was suggested in a previous study to be a factor as well, based on the observation that incremental changes in PTPase loop structure by single point mutations to alanine often induce incremental changes in enzyme catalytic efficiency. In this study, the temperature jump relaxation spectroscopy (T-jump) has been used to discern the subtle changes of the loop dynamics due to point loop mutations. As expected, our results suggest a correlation between loop dynamics and the size of the residue on the catalytic loop. The stabilization of the enzyme-ligand complex is often enthalpy driven, achieved by formation of additional favorable hydrogen bonding/ionic interactions after loop closure. Interestingly, our T-jump and X-ray crystallography studies on YopH suggest that the elimination of some ligand-protein interactions by mutation does not necessarily destabilize the ligand-enzyme complex after loop closure, since the increased entropy in the forms of more mobile protein residues may be sufficient to compensate the free energy loss due to lost interactions and may even lead to enhanced efficiency of the enzyme catalysis. How these competing loop properties may affect loop dynamics and enzyme function are discussed.

Inhibition of Yersinia protein tyrosine phosphatase by phosphonate derivatives of calixarenes

Inhibition of Yersinia protein tyrosine phosphatase by calix[4]arene mono-, bis-, and tetrakis(methylenebisphosphonic) acids as well as calix[4]arene and thiacalix[4]arene tetrakis(methylphosphonic) acids have been investigated. The kinetic studies revealed that some compounds in this class are potent competitive inhibitors of Yersinia PTP with inhibition constants in the low micromolar range. The binding modes of macrocyclic phosphonate derivatives in the enzyme active center have been explained using computational docking approach. The results obtained indicate that calix[4]arenes are promising scaffolds for the development of inhibitors of Yersinia PTP.

Impaired acid catalysis by mutation of a protein loop hinge residue in a YopH mutant revealed by crystal structures.

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 A structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO](2) core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

A Mining Minima Approach to Exploring the Docking Pathways of p-Nitrocatechol Sulfate to YopH

Using the docking of p-nitrocatechol sulfate to Yersinia protein tyrosine phosphatase YopH as an example, we showed that an approach based on mining minima followed by cluster and similarity analysis could generate useful insights into docking pathways. Our simulation treated both the ligand and the protein as flexible molecules so that the coupling between their motion could be properly accounted for. Our simulation identified three docking poses; the one with the lowest energy agreed well with experimental structure. The model also predicted the side-chain conformations of the amino acids lying in the binding pocket correctly with the exception of three residues that appeared to be stabilized by two structural water molecules in the crystal structure. The implicit solvent model employed in the simulation could not capture such effects well. We also found four major pathways leading to these docking poses after the ligand entered the mouth of the binding pocket. In addition, the sulfate group of p-nitrocatechol sulfate was found to be important both in binding the ligand to the pocket and in guiding the ligand to dock into the pocket. The coupling of the motion between the protein and the ligand also played an important role in facilitating ligand loading and unloading.

Loop Dynamics and Ligand Binding Kinetics in the Reaction Catalyzed by the Yersinia Protein Tyrosine Phosphatase

The Yersinia protein tyrosine phosphatase (YopH) contains a loop of ten amino acids (the WPD loop) that covers the entrance of the active site of the enzyme during substrate binding. In this work the substrate mimicking competitive inhibitor p-nitrocatechol sulfate (PNC) is used as a probe of the active site. The dynamics of the WPD loop was determined by subjecting an equilibrated system containing YopH, PNC, and YopH bound to PNC to a laser induced temperature jump, and subsequently following the change in equilibrium due to the perturbation. Using this methodology the dynamics associated with substrate binding in YopH have been determined. These results indicate that substrate binding is coupled to the WPD loop motion, and WPD loop dynamics occur in the sub-millisecond time scale. The significance of these dynamic results is interpreted in terms of the catalytic cycle of the enzyme.

A two stage click-based library of protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type 2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed ‘click’ cycloaddition reactions between azides and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoylformate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting α-ketocarboxylic acids were assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one compound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first generation library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and evaluated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR, and CD45. The best bis(α-ketocarboxylic acid) inhibitor 34 had an IC 50 value of 550 nM against the Yersinia PTP and an IC 50 value of 710 nM against TCPTP. The most potent inhibitor containing a single α-ketocarboxylic acid group 32 had IC 50 values of 2.1, 5.7, and 2.6 μM against the Yersinia PTP, PTP1B, and TCPTP, respectively.

Dynamics of the WPD Loop of the Yersinia Protein Tyrosine Phosphatase

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia spp., causing gastrointestinal diseases and the plague. Like eukaryotic PTPases, YopH catalyzes the hydrolysis of the phosphate moiety of phosphotyrosine within a highly conserved binding pocket, which is also characterized by the closure of the so-called “WPD loop” upon ligand binding. In this study, we investigate the conformational changes and dynamics of the WPD loop by molecular dynamics simulations. Consistent with experimental observations, our simulations show that the WPD loop of YopH is intrinsically flexible and fluctuates between the open and closed conformation with a frequency of ∼4 ns for the apo, native protein. The region of helix α4 spanning loop 384-392, which has been revealed experimentally as a second substrate-binding site in YopH, is found to be highly associated with the WPD loop, stabilizing it in the closed, active conformation, and providing a structural basis for the cooperation of the second-substrate binding site in substrate recognition. Loop L4 (residues 323–327) is shown to be involved in a parallel, correlated motion mode with the WPD loop that contributes the stabilization of a more extended open conformation. In addition, we have simulated the loop reopening in the ligand-bound protein complex by applying the locally enhanced sampling method. Finally, the dynamic behavior of the WPD loop for the C403S mutant differs from the wild-type YopH remarkably. These results shed light on the role of the WPD loop in PTPase-mediated catalysis, and are useful in structure-based design for novel, selective YopH inhibitors as antibacterial drugs.

Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

An assay method that continuously measures the protein tyrosine phosphatase (PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energy transfer (FRET) was developed as an improvement of our previously reported discontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A. Matsumoto, Biochem. J. 343 (1999) 385–391]. The assay uses oligopeptide substrates that contain (7-methoxycoumarin-4-yl)acetyl (Mca) group as a fluorescence donor and 2,4-dinitrophenyl (DNP) group as a fluorescence acceptor, in addition to a phosphotyrosine residue located between these two groups. In the assay, a PTP solution is added to a buffer solution containing a FRET substrate and chymotrypsin. The PTP-catalyzed dephosphorylation of the substrate and subsequent chymotryptic cleavage of the dephosphorylated substrate results in a disruption of FRET, thereby increasing Mca fluorescence. In this study, we used FRET substrates that are much more susceptible to chymotryptic cleavage after dephosphorylation than the substrate used in our discontinuous assay, thus enabling the continuous assay without significant PTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflected the rate of dephosphorylation at appropriate chymotrypsin concentrations. Since the continuous assay allows the measurement of initial rate of dephosphorylation reaction, kinetic parameters for the dephosphorylation reactions of FRET substrates by Yersinia, T-cell and LAR PTPs were determined. The continuous assay was compatible with the measurement of very low PTP activity in a crude enzyme preparation and was comparable in sensitivity to assays that use radiolabeled substrates.

Proteomic and functional analysis of the suite of Ysp proteins exported by the Ysa type III secretion system of Yersinia enterocolitica Biovar 1B

Full virulence of Yersinia enterocolitica Biovar 1B requires two distinct and distantly related contact-dependent type III secretion (T3S) systems. The plasmid-encoded Ysc T3S system is essential for systemic stages of infection and the Yop effector proteins it translocates have been extensively studied. The chromosome-encoded Ysa T3S system contributes to gastrointestinal stages of infection, but the suite of Ysp effectors proteins it translocates into host cells remains obscure. Using a proteomics-based approach, the Ysa T3S system was analysed revealing a complex set of 15 secreted Ysp proteins. Seven of these proteins were previously described (YspA, YspB, YspC, YspD, YopE, YopN and YopP). Eight of these Ysps (YspK, YspI, YspE, YspF, YspP, YspY, YspN and YspL) had not previously been characterized. Several of the new Ysps are homologous to other virulence factors, including YspP with similarity to the Yersinia protein tyrosine phosphatase YopH and YspK with similarity to the Shigella serine/threonine kinase OspG. Biochemical analysis of purified hexa-histidine tagged YspK and YspP established that these proteins have kinase and phosphatase activity respectively. Infection of eukaryotic cells with Y. enterocolitica strains expressing a Ysp-CyaA chimeric protein resulted in Ysa T3S system-dependent increases in cytosolic levels of cAMP for six Ysps (YspK, YspI, YspE, YspF, YspP and YspL), but not two others (YspY and YspN). YspN, however, was required for translocation of effector proteins into eukaryotic cells by the Ysa T3S system. Competition assays in BALB/c mice revealed that mutants defective for the production of an individual Ysp are affected for colonization of gastrointestinal tissues. Collectively, the results of this study support the hypothesis that the Ysa T3S system targets a complex suite of effector proteins into host cells to affect the outcome of an infection. Identification of the suite of effectors delivered by the Ysa T3S system reveals that host cell signalling pathways are the probable targets of several Ysp effectors.

Synthesis of tripeptides as potent Yersinia protein tyrosine phosphatase inhibitors

We report the synthesis of a series of monoanionic phosphotyrosyl (pTyr) mimetic-containing tripeptides based on ‘Fmoc-Glu(OBn)-Xxx-Leu-amide’ (where Xxx = pTyr mimetic) and their N-terminally modified derivatives. The inhibitory potencies of compounds were tested against YopH and human PTP1B enzymes. Several compounds exhibited noteworthy activity against both YopH and PTP1B. Among the N-terminally modified analogues, 5-methylindole derivative 30 was found to be the best moiety to replace base-labile Fmoc group. A mode of binding with YopH is proposed for tripeptides 21, 30, and 31.

Molecular docking and 3D-QSAR studies of Yersinia protein tyrosine phosphatase YopH inhibitors

Three-dimensional quantitative structure–activity relationship (QSAR) studies were conducted on two classes of recently explored compounds with known YopH inhibitory activities. Docking studies were employed to position the inhibitors into the YopH active site to determine the probable binding conformation. Good correlations between the predicated binding free energies and the inhibitory activities were found for two subsets of phosphate mimetics: α-ketocarboxylic acid and squaric acid ( R 2 = 0.70 and 0.68, respectively). The docking results also provided a reliable conformational alignment scheme for 3D-QSAR modeling. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed based on the docking conformations, giving q 2 of 0.734 and 0.754 for CoMFA and CoMSIA models, respectively. The 3D-QSAR models were significantly improved after removal of an outlier ( q 2 = 0.829 for CoMFA and q 2 = 0.837 for CoMSIA). The predictive ability of the models was validated using a set of compounds that were not included in the training set. Mapping the 3D-QSAR models to the active site of YopH provides new insight into the protein–inhibitor interactions for this enzyme. These results should be applicable to the prediction of the activities of new YopH inhibitors, as well as providing structural implications for designing potent and selective YopH inhibitors as antiplague agents.

Suramin Derivatives as Inhibitors and Activators of Protein-tyrosine Phosphatases

Protein-tyrosine phosphatases (PTPs) are important signaling enzymes that have emerged within the last decade as a new class of drug targets. It has previously been shown that suramin is a potent, reversible, and competitive inhibitor of PTP1B and Yersinia PTP (YopH). We therefore screened 45 suramin analogs against a panel of seven PTPs, including PTP1B, YopH, CD45, Cdc25A, VHR, PTPalpha, and LAR, to identify compounds with improved potency and specificity. Of the 45 compounds, we found 11 to have inhibitory potency comparable or significantly improved relative to suramin. We also found suramin to be a potent inhibitor (IC(50) = 1.5 microm) of Cdc25A, a phosphatase that mediates cell cycle progression and a potential target for cancer therapy. In addition we also found three other compounds, NF201, NF336, and NF339, to be potent (IC(50) < 5 microm) and specific (at least 20-30-fold specificity with respect to the other human PTPs tested) inhibitors of Cdc25A. Significantly, we found two potent and specific inhibitors, NF250 and NF290, for YopH, the phosphatase that is an essential virulence factor for bubonic plague. Two of the compounds tested, NF504 and NF506, had significantly improved potency as PTP inhibitors for all phosphatases tested except for LAR and PTPalpha. Surprisingly, we found that a significant number of these compounds activated the receptor-like phosphatases, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane-distal cytoplasmic PTP domain (D2) of PTPalpha: the direct intramolecular regulation of the activity of the membrane-proximal cytoplasmic PTP domain (D1). Binding of certain of these compounds to PTPalpha disrupts D1-D2 basal state contacts and allows new contacts to occur between D1 and D2, which activates D1 by as much as 12-14-fold when these contacts are optimized.

Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

Chemical and Mechanistic Approaches to the Study of Protein Tyrosine Phosphatases

AbstractFor Abstract see ChemInform Abstract in Full Text.

Crystal Structure of the Yersinia Protein-tyrosine Phosphatase YopH Complexed with a Specific Small Molecule Inhibitor

The pathogenic bacteria Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to bubonic plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a Ki value of 25 microM for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-A resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors.

Tripeptide inhibitors of Yersinia protein-tyrosine phosphatase.

The protein-tyrosine phosphatase (PTP) 'YopH' is a virulence factor of Yersinia pestis, the causative agent of plague. Potential use of Yersinia as a bioterrorism agent renders YopH inhibitors of therapeutic importance. Previously, we had examined the inhibitory potencies of a variety of phosphotyrosyl (pTyr) mimetics against the human PTP1B enzyme by displaying them in the EGFR-derived hexapeptide sequence, 'Ac-Asp-Ala-Asp-Glu-Xxx-Leu-amide', where Xxx=pTyr mimetic. The poor inhibitory potencies of certain of these pTyr mimetics were attributed to restricted orientation within the PTP1B catalytic pocket incurred by extensive peripheral interaction of the hexapeptide platform. Utilizing the smaller tripeptide platform, 'Fmoc-Glu-Xxx-Leu-amide' we demonstrate herein that several of the low affinity hexapeptide-expressed pTyr mimetics exhibit high PTP1B affinity within the context of the tripeptide platform. Of particular note, the mono-anionic 4-(carboxydifluoromethyl)Phe residue exhibits affinity equivalent to the di-anionic F(2)Pmp residue, which had previously been among the most potent PTP-binding motifs. Against YopH, it was found that all tripeptides having Glu residues with an unprotected side chain carboxyl were inactive. Alternatively, in their Glu-OBn ester forms, several of the tripeptides exhibited good YopH affinity with the mono-anionic peptide, Fmoc-Glu(OBn)-Xxx-Leu-amide, where Xxx=4-(carboxymethyloxy)Phe providing an IC(50) value of 2.8 microM. One concern with such inhibitors is that they may potentially function by non-specific mechanisms. Studies with representative inhibitors, while failing to provide evidence of a non-specific promiscuous mode of inhibition, did indicate that non-classical inhibition may be involved.