The apo-structure of the low molecular weight protein-tyrosine phosphatase A (MptpA) from Mycobacterium tuberculosis allows for better target-specific drug development.
ABSTRACT: Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX(5)R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ?10 ?, from an "open" to a "closed" conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.
Project description:A putative low molecular weight protein tyrosine phosphatase (LMW-PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high-resolution NMR spectroscopy using NOE-derived distance restraints, hydrogen bond data, and torsion angle restraints. The three-dimensional structure consists of a central four-stranded parallel beta-sheet flanked by five alpha-helices, revealing an overall structural topology similar to those of the eukaryotic LMW-PTPs, such as human HCPTP-A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW-PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR-based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2-hydroxy-5-nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three-dimensional structure and ligand-binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three-dimensional structure along with mapping of small-molecule binding will be discussed in the context of developing high-affinity inhibitors of this novel LMW-PTP.
Project description:The low-molecular-weight (LMW) protein tyrosine phosphatases (PTPs) exist ubiquitously in prokaryotes and eukaryotes and play important roles in cellular processes. We report here the solution structure of YwlE, an LMW PTP identified from the gram-positive bacteria Bacillus subtilis. YwlE consists of a twisted central four-stranded parallel beta-sheet with seven alpha-helices packing on both sides. Similar to LMW PTPs from other organisms, the conformation of the YwlE active site is favorable for phosphotyrosine binding, indicating that it may share a common catalytic mechanism in the hydrolysis of phosphate on tyrosine residue in proteins. Though the overall structure resembles that of the eukaryotic LMW PTPs, significant differences were observed around the active site. Residue Asp115 is likely interacting with residue Arg13 through electrostatic interaction or hydrogen bond interaction to stabilize the conformation of the active cavity, which may be a unique character of bacterial LMW PTPs. Residues in the loop region from Phe40 to Thr48 forming a wall of the active cavity are more flexible than those in other regions. Ala41 and Gly45 are located near the active cavity and form a noncharged surface around it. These unique properties demonstrate that this loop may be involved in interaction with specific substrates. In addition, the results from spin relaxation experiments elucidate further insights into the mobility of the active site. The solution structure in combination with the backbone dynamics provides insights into the mechanism of substrate specificity of bacterial LMW PTPs.
Project description:The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a regulator of a number of signaling pathways and has been implicated as a potential target for oncology and diabetes/obesity. There is significant therapeutic interest in developing potent and selective inhibitors to control LMW-PTP activity. We report the discovery of a novel class of LMW-PTP inhibitors derived from sulfophenyl acetic amide (SPAA), some of which exhibit greater than 50-fold preference for LMW-PTP over a large panel of PTPs. X-ray crystallography reveals that binding of SPAA-based inhibitors induces a striking conformational change in the LMW-PTP active site, leading to the formation of a previously undisclosed hydrophobic pocket to accommodate the ?-phenyl ring in the ligand. This induced-fit mechanism is likely a major contributor responsible for the exquisite inhibitor selectivity.
Project description:The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.
Project description:Tuberculosis (TB) is a leading cause of death worldwide and its impact has intensified due to the emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. Protein phosphorylation plays a vital role in the virulence of Mycobacterium tuberculosis (M.tb) mediated by protein kinases. Protein tyrosine phosphatase A (MptpA) undergoes phosphorylation by a unique tyrosine-specific kinase, protein tyrosine kinase A (PtkA), identified in the M.tb genome. PtkA phosphorylates PtpA on the tyrosine residues at positions 128 and 129, thereby increasing PtpA activity and promoting pathogenicity of MptpA. In the present study, we performed an extensive investigation of the conformational behavior of the intrinsically disordered domain (IDD) of PtkA using replica exchange molecular dynamics simulations. Long-term molecular dynamics (MD) simulations were performed to elucidate the role of IDD on the catalytic activity of kinase core domain (KCD) of PtkA. This was followed by identification of the probable inhibitors of PtkA using drug repurposing to block the PtpA-PtkA interaction. The inhibitory role of IDD on KCD has already been established; however, various analyses conducted in the present study showed that IDDPtkA had a greater inhibitory effect on the catalytic activity of KCDPtkA in the presence of the drugs esculin and inosine pranobex. The binding of drugs to PtkA resulted in formation of stable complexes, indicating that these two drugs are potentially useful as inhibitors of M.tb.
Project description:Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate.
Project description:Dysregulation of protein tyrosine phosphorylation has been implicated in a number of human diseases, including cancer, diabetes, and neurodegenerative diseases. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here we report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, striatal-enriched protein tyrosine phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their respective targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concentrations. Additionally, active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry, and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.
Project description:LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date and was originally misclassified as an atypical dual-specificity phosphatase. Kinetic isotope effects with steady-state and pre-steady-state kinetics of VHZ and mutants with p-nitrophenol phosphate have revealed several unusual properties. VHZ is significantly more active than previously reported but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations of Q-loop residues affect this phosphotransferase activity, mutations on the IPD loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer.
Project description:Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.
Project description:Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.