Structural basis for selective inhibition of Mycobacterium tuberculosis protein tyrosine phosphatase PtpB

Tyrosine kinases and phosphatases establish the crucial balance of tyrosine phosphorylation in cellular signaling, but creating specific inhibitors of protein Tyr phosphatases (PTPs) remains a challenge. Here, we report the development of a potent, selective inhibitor of Mycobacterium tuberculosis PtpB, a bacterial PTP that is secreted into host cells where it disrupts unidentified signaling pathways. The inhibitor, (oxalylamino-methylene)-thiophene sulfonamide (OMTS), showed an IC(50) of 440 +/- 50 nM and >60-fold specificity for PtpB over six human PTPs. The 2 A resolution crystal structure of PtpB in complex with OMTS revealed a large rearrangement of the enzyme, with some residues shifting >27 A relative to the PtpB:PO(4) complex. Extensive contacts with the catalytic loop provide a potential basis for inhibitor selectivity. Two OMTS molecules bound adjacent to each other, raising the possibility of a second substrate phosphotyrosine binding site in PtpB. The PtpB:OMTS structure provides an unanticipated framework to guide inhibitor improvement.     

Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.     

Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site

Ribose-5-phosphate isomerases (EC 5.3.1.6) inter-convert ribose-5-phosphate and ribulose-5-phosphate. This reaction allows the synthesis of ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.     

Synthetic chalcones as efficient inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

In the search for lead compounds for new drugs for tuberculosis, the activity of 38 synthetic chalcones were assayed for their potential inhibitory action towards a protein tyrosine phosphatase from Mycobacterium tuberculosis--PtpA. The compounds were obtained by aldolic condensation between aldehydes and acetophenones, under basic conditions. Five compounds presented moderate or good activity. The structure-activity analysis reveals that the predominant factor for the activity is the molecule planarity/hydrophobicity and the nature of the substituents.     

Crystal structure of human riboflavin kinase reveals a beta barrel fold and a novel active site arch

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B(2)) to form FMN, an obligatory step in vitamin B(2) utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel beta barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3(10) helix, and a reverse turn followed by a short beta strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.     

Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K_i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure–activity relationships observed in vitro and revealed further possibilities for compound development.     

Alpha-methylacyl-CoA racemase from Mycobacterium tuberculosis. Mutational and structural characterization of the active site and the fold

Alpha-methylacyl-CoA racemase (Amacr) catalyzes the racemization of alpha-methyl-branched CoA esters. Sequence comparisons have shown that this enzyme is a member of the family III CoA transferases. The mammalian Amacr is involved in bile acid synthesis and branched-chain fatty acid degradation. In human, mutated variants of Amacr have been shown to be associated with disease states. Amino acid sequence alignment of Amacrs and its homologues from various species revealed 26 conserved protic residues, assumed to be potential candidates as catalytic residues. Amacr from Mycobacterium tuberculosis (MCR) was taken as a representative of the racemases. To determine their importance for efficient catalysis, each of these 26 protic residues of MCR was mutated into an alanine, respectively, and the mutated variants were overexpressed in Escherichia coli. It was found that four variants (R91A, H126A, D156A, and E241A) were properly folded but had much decreased catalytic efficiency. Apparently, Arg91, His126, Asp156, and Glu241 are important catalytic residues of MCR. The importance of these residues for catalysis can be rationalized by the 1.8 A resolution crystal structure of MCR, which shows that the catalytic site is at the interface between the large and small domain of two different subunits of the dimeric enzyme. This crystal structure is the first structure of a complete enzyme of the bile acid synthesis pathway. It shows that MCR has unique structural features, not seen in the structures of the sequence related formyl-CoA transferases, suggesting that the family III CoA transferases can be subdivided in at least two classes, being racemases and CoA transferases.     

The structure of the bovine protein tyrosine phosphatase dimer reveals a potential self-regulation mechanism.

The bovine protein tyrosine phosphatase (BPTP) is a member of the class of low-molecular weight protein tyrosine phosphatases (PTPases) found to be ubiquitous in mammalian cells. The catalytic site of BPTP contains a CX(5)R(S/T) phosphate-binding motif or P-loop (residues 12-19) which is the signature sequence for all PTPases. Ser19, the final residue of the P-loop motif, interacts with the catalytic Cys12 and participates in stabilizing the conformation of the active site through interactions with Asn15, also in the P-loop. Mutations at Ser19 result in an enzyme with altered kinetic properties with changes in the pK(a) of the neighboring His72. The X-ray structure of the S19A mutant enzyme shows that the general conformation of the P-loop is preserved. However, changes in the loop containing His72 result in a displacement of the His72 side chain that may explain the shift in the pK(a). In addition, it was found that in the crystal, the protein forms a dimer in which Tyr131 and Tyr132 from one monomer insert into the active site of the other monomer, suggesting a dual-tyrosine motif on target sites for this enzyme. Since the activity of this PTPase is reportedly regulated by phosphorylation at Tyr131 and Tyr132, the structure of this dimer may provide a model of a self-regulation mechanism for the low-molecular weight PTPases.     

Analyzing the catalytic mechanism of protein tyrosine phosphatase PtpB from Staphylococcus aureus through site-directed mutagenesis

Protein tyrosine phosphatase B (PtpB) from Staphylococcus aureus, MRSA 252, is a low molecular weight protein tyrosine phosphatase involved in its pathogenicity. PtpB has been modeled in silico and site-directed mutagenesis performed to ascertain the importance of active site residues Cys8, Arg14, Ser15 and Asp120 in its catalytic mechanism. Kinetic characterization of wild-type and the mutant PtpBs, C8S, R14A, S15T, S15A, D120A, D120E, D120N revealed the reaction mechanism followed by this LMWPTPase. The mutations caused major changes in the local environment resulting in significant decrease of its catalytic activity. Inhibition kinetics for the wild-type enzyme was performed with maleimide and maleimidobutyric acid.     

Active site labeling of a receptor-like protein tyrosine phosphatase.

The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine.     

Theoretical studies on the interaction of biphenyl inhibitors with Mycobacterium tuberculosis protein tyrosine phosphatase MptpB

MptpB is an essential secreted virulence factor for M. tuberculosis. Inhibition of MptpB impairs mycobacterial survival in host macrophages and thus helps reduce tuberculosis infections. However, the binding mode of the biphenyl inhibitors, which are known as some of the most potent MptpB inhibitors, remains unclear. In this study, to understand the interactions between biphenyl inhibitors and MptpB, docking and molecular dynamics simulations were carried out using AutoDock and GROMACS softwares. Calculation results show that all the biphenyl inhibitors can be docked to the binding site of MptpB, with the acid warheads forming a hydrogen bond network at the active site. But the binding modes of other terminals of these inhibitors are different. The cyclohexyl and trifluoromethyl substituents at R1 and R2 sites are necessary for the inhibitors to adopt their double-site binding mechanism. The estimated binding affinities are basically consistent with the experimental results. MD simulations show that these binding complexes display different stability.     

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.     

Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a β-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The synthesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain of CPs with the enzyme active site residues. All these are well supported by docking studies.     

Solution structure of a low-molecular-weight protein tyrosine phosphatase from Bacillus subtilis

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.     

Protein tyrosine phosphatase PtpA is not required for Mycobacterium tuberculosis growth in mice

Mycobacterium tuberculosis (Mtb) alters the host response to infection by secreting protein factors. Mtb produces two secreted protein tyrosine phosphatases, PtpA and PtpB, which are thought to interfere with host signaling. Deletion of ptpA or ptpB attenuates bacterial growth in activated macrophages. To address the in vivo function of PtpA, we generated a genetic deletion mutant, DeltaptpA. The mutant was not defective when grown in vitro, consistent with the presumed role of PtpA in the host. The ptpA mutant, however, also showed no growth defect in a mouse infection model. The absence of a growth defect in mice suggests that the requirement for PtpA differs in mouse and human infections, and that mice are not a suitable infection model for the study of PtpA.     

NMR solution structure of the archaebacterial chromosomal protein MC1 reveals a new protein fold

The three-dimensional structure of methanogen chromosomal protein 1 (MC1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55, has been solved using (1)H NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an alpha helix and five beta strands, arranged as two antiparallel beta sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two beta sheets and the helix side facing the interior of the protein. The MC1 solution structure shows a globular protein with overall dimensions in the range of 34-40 A, which potentially corresponds to a DNA-binding site of 10-12 base pairs. The presumed DNA-binding site is located on the sequence comprising residues K62-P82, which is formed by a part of strands II2 and II3 belonging to the triple-stranded antiparallel beta sheet and a loop flanked by prolines P68 and P76. The tryptophan W74 that is expected to play a key role in the DNA-binding according to photocross-linking experiments was found completely exposed to the solvent, in a good position to interact with DNA. The overall fold of MC1, characterized by its linking beta-beta-alpha-beta-beta-loop-beta, is different from other known DNA-binding proteins. Its structure suggests a different DNA-binding mode than those of the histone-like proteins HU or HMGB. Thus, MC1 may be classified as a member of a new family.     

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.     

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.     

The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis

Mycobacterium tuberculosis, the cause of tuberculosis, is a devastating human pathogen. The emergence of multidrug resistance in recent years has prompted a search for new drug targets and for a better understanding of mechanisms of resistance. Here we focus on the gene product of an open reading frame from M. tuberculosis, Rv1347c, which is annotated as a putative aminoglycoside N-acetyltransferase. The Rv1347c protein does not show this activity, however, and we show from its crystal structure, coupled with functional and bioinformatic data, that its most likely role is in the biosynthesis of mycobactin, the M. tuberculosis siderophore. The crystal structure of Rv1347c was determined by multiwavelength anomalous diffraction phasing from selenomethionine-substituted protein and refined at 2.2 angstrom resolution (r = 0.227, R(free) = 0.257). The protein is monomeric, with a fold that places it in the GCN5-related N-acetyltransferase (GNAT) family of acyltransferases. Features of the structure are an acyl-CoA binding site that is shared with other GNAT family members and an adjacent hydrophobic channel leading to the surface that could accommodate long-chain acyl groups. Modeling the postulated substrate, the N(epsilon)-hydroxylysine side chain of mycobactin, into the acceptor substrate binding groove identifies two residues at the active site, His130 and Asp168, that have putative roles in substrate binding and catalysis.