Protein Tyrosine Phosphatase YopH 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.

Loop Dynamics and Enzyme Catalysis in Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) play an important role in cellular signaling 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 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 neighboring 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.

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.

Conservative tryptophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-loop function and crystallize with divanadate esters in their active sites.

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution.

A new class of salicylic acid derivatives for inhibiting YopH of Yersinia pestis

Previously, we identified a class of salicylic acid derivatives that display inhibitory activity against the protein tyrosine phosphatase YopH from Yersinia pestis. Because docking study suggested that the large phenyl ring attaching to the salicylic acid core might be exposed to the solvent and might not contribute significantly to binding, we have developed a new class of compounds that no longer contain this phenyl ring. We first devised a synthetic scheme for the compounds and then developed an automated computational screening model surrounding this synthetic scheme to help select a small number of compounds for synthesis and experimental testing. Based on this computational screening model and the analysis of the structure–activity relationship of our previous class of compounds, we have synthesized eight compounds and found five that yield micromolar activity. When applying in a larger scale, the synthetic scheme and the computational screening model developed here should help to identify even more potent inhibitors in the future.

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.

The phosphoramidase competency of prototypical phosphatase motifs

The discovery that phosphorylation of proteins occurs on nitrogen by particular kinases raises the question of whether a separate class of phosphoramidases also exists, or, if known phosphatases carry out the hydrolysis of phosphoramidates. The phosphoramidase activity of a number of phosphatases with different catalytic motifs was studied using the substrates N‐phenylphosphoramidate (N‐PhPAM) and phosphoryl imidazole (PIm). The phosphatases assayed were: the protein tyrosine phosphatase YopH; alkaline phosphatase; the dual‐specificity phosphatase VHR; PHPT1, the only known phosphohistidine phosphatase; and, the serine/threonine phosphatase PP1. The catalytic efficiencies, kcat/Km (s−1M−1), were compared for the respective phosphoramidase and phosphatase activities for each enzyme. Ratios of catalytic efficiencies (kcat/Km)/(kcat/Km) of phosphoester (pNPP or phenyl phosphate) over PIm are: YopH – 43; AP – 4.1; VHR – 0.23; and PHPT1 – 0.0065. PP1 exhibited no phosphoramidase activity. The results show that most phosphatase catalytic motifs display catalytic promiscuity by cleaving both phosphoesters and phosphoramidates, but with a pronounced preference for one substrate type versus the other. (Funding from NIH GM47297)

Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH.

Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, asexemplified by the structures of several PTP1B-inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing ahighly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors.

Design and NMR studies of cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH.

We report on the design and evaluation of novel cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH from Yersinia. Cyclic peptides have been designed based on a short sequence from the protein SKAP-HOM [DE(pY)DDPF (pY=phosphotyrosine)], and they all contain the motif DEZXDPfK (where Z is a phosphotyrosine or a non-hydrolyzable phosphotyrosine mimetic, X is an aspartic acid or a leucine and f is a d-phenylalanine). These peptides present a 'head to tail' architecture, enabling cyclization through formation of an amide bond in between the side chains of the first aspartic acid and the lysine residues. Chemical shift perturbation studies have been carried out to monitor the binding of these peptides to the N-terminal domain of YopH. Peptides containing a phosphotyrosine moiety exhibit binding affinities in the low micromolar range; substitution of the phosphotyrosine with one of its non-hydrolyzable derivatives dramatically reduces the binding affinities. These preliminary studies may pave the way for the discovery of more potent and selective peptide-based ligands of the YopH N-terminal domain which could be further investigated for their ability to inhibit Yersiniae infections.

Derivatives of Salicylic Acid as Inhibitors of YopH in Yersinia pestis

Yersinia pestis causes diseases ranging from gastrointestinal syndromes to bubonic plague and could be misused as a biological weapon. As its protein tyrosine phosphatase YopH has already been demonstrated as a potential drug target, we have developed two series of forty salicylic acid derivatives and found sixteen to have micromolar inhibitory activity. We designed these ligands to have two chemical moieties connected by a flexible hydrocarbon linker to target two pockets in the active site of the protein to achieve binding affinity and selectivity. One moiety possessed the salicylic acid core intending to target the phosphotyrosine-binding pocket. The other moiety contained different chemical fragments meant to target a nearby secondary pocket. The two series of compounds differed by having hydrocarbon linkers with different lengths. Before experimental co-crystal structures are available, we have performed molecular docking to predict how these compounds might bind to the protein and to generate structural models for performing binding affinity calculation to aid future optimization of these series of compounds.

NMR-based design and evaluation of novel bidentate inhibitors of the protein tyrosine phosphatase YopH.

We describe the use of a furanyl salicyl nitroxide derivative ('spin-labeled' compound), as a paramagnetic phosphotyrosine mimetic, to carry out a second-site screening by NMR against the PTPase YopH from Yersinia pestis. Using such a fragment-based screening approach we identified several small molecules targeting YopH that bind at sites adjacent to the spin-labeled compound. These second-site fragments were subsequently used to design and synthesize bidentate YopH inhibitors with submicromolar in vitro inhibition, selectivity against the human PTPase PTP1B, and cellular activity against Y. pseudotuberculosis. These initial compounds could result useful in elucidating the structural determinants necessary for YopH inhibition and may help in the design of even more active, selective and cell permeable compounds for the development of novel therapies against Yersiniae.

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.

Genetic differences between blight-causing Erwinia species with differing host specificities, identified by suppression subtractive hybridization.

PCR-based subtractive hybridization was used to isolate sequences from Erwinia amylovora strain Ea110, which is pathogenic on apples and pears, that were not present in three closely related strains with differing host specificities: E. amylovora MR1, which is pathogenic only on Rubus spp.; Erwinia pyrifoliae Ep1/96, the causal agent of shoot blight of Asian pears; and Erwinia sp. strain Ejp556, the causal agent of bacterial shoot blight of pear in Japan. In total, six subtractive libraries were constructed and analyzed. Recovered sequences included type III secretion components, hypothetical membrane proteins, and ATP-binding proteins. In addition, we identified an Ea110-specific sequence with homology to a type III secretion apparatus component of the insect endosymbiont Sodalis glossinidius, as well as an Ep1/96-specific sequence with homology to the Yersinia pestis effector protein tyrosine phosphatase YopH.

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.

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.

Interaction between the Yersinia Tyrosine Phosphatase YopH and Its Macrophage Substrate, Fyn-Binding Protein, Fyb

Pathogenic Yersinia species can evade phagocytosis by injecting virulence effectors that interfere with the phagocytic machinery of host cells. One of these virulence effectors is the protein tyrosine phosphatase YopH. Through its enzymatic activity, YopH interferes with the initial phagocytic process by affecting signalling for cytoskeletal rearrangements. Fyb (Fyn-binding protein), which is an immune cell-specific adaptor protein, has been identified as a substrate of YopH in macrophages. In this study, the interaction between YopH and Fyb is studied. We show that YopH binds to Fyb via different regions in both phosphotyrosine-dependent and phosphotyrosine-independent ways. The phosphotyrosine substrate binding N-terminal part (1–130) of YopH as well as the C-terminal catalytic region binds to Fyb in a phosphotyrosine-dependent manner. We also show that a central part of YopH (130–260) interacts with the Fyb C-terminus (548–783) in a phosphotyrosine-independent manner. Further, we demonstrate that the N-terminal binding region of YopH is important for YopH-mediated functions on macrophages such as dephosphorylation of Fyb, blockage of phagocytosis, and cytotoxic effects.