RESUMO
Cyclic dinucleotides (CDNs) are a significant and expanding class of secondary messengers that influence several vital bacterial physiological functions. Therefore, an understanding of the process by which CDNs are degraded by their cognate PDEs is crucial for comprehending a variety of cellular processes, such as the formation and dissemination of biofilms. As an alternative, it might be beneficial to create and/or identify non-hydrolyzable CDN derivatives to employ them as chemical probes of cyclic-di-GMP (c-di-GMP) signaling. Cyclic-di-inosine monophosphate, or c-di-IMP, is not a naturally occurring signaling molecule in biological systems, but it has strong adjuvant effects on metazoans and functions as an immunological modulator and stimulant. Here we report the first structural details of c-di-IMP and EAL interaction through high-resolution (2.2 Å) crystal structure of VcEAL in complex with c-di-IMP + Ca2+. Comparison of the VcEAL structures bound with cyclic-di-GMP (c-di-GMP), 3',5'-cyclic-AMP-GMP (cGAMP) and c-di-IMP and the structural variations at the chemical level between these CDNs provides their structural basis of recognition and rate of hydrolysis.
RESUMO
VpsR, the master regulator of biofilm formation in Vibrio cholerae, is an atypical NtrC1 type bEBP lacking residues essential for σ54-RNAP binding and REC domain phosphorylation. Moreover, transcription from PvpsL, a promoter of biofilm biosynthesis, has been documented in presence of σ70-RNAP/VpsR/c-di-GMP complex. It was proposed that c-di-GMP and VpsR together form an active transcription complex with σ70-RNAP. However, the impact of c-di-GMP imparted on VpsR that leads to transcription activation with σ70-RNAP remained elusive, largely due to the lack of the structure of VpsR and knowledge about c-di-GMP:VpsR interactions. In this direction we have solved the crystal structure of VpsRRA, containing REC and AAA+ domains, in apo, AMPPNP/GMPPNP and c-di-GMP bound states. Structures of VpsRRA unveiled distinctive REC domain orientation that leads to a novel dimeric association and noncanonical ATP/GTP binding. Moreover, we have demonstrated that at physiological pH VpsR remains as monomer having no ATPase activity but c-di-GMP imparted cooperativity to convert it to dimer with potent activity. Crystal structure of c-di-GMP:VpsRRA complex reveals that c-di-GMP binds near the C-terminal end of AAA+ domain. Trp quenching studies on VpsRR, VpsRA, VpsRRA, VpsRAD with c-di-GMP additionally demonstrated that c-di-GMP could potentially bind VpsRD. We propose that c-di-GMP mediated tethering of VpsRD with VpsRA could likely favor generating the specific protein-DNA architecture for transcription activation.
Assuntos
Adenosina Trifosfatases/metabolismo , Sítios de Ligação , GMP Cíclico/análogos & derivados , DNA Bacteriano/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/metabolismo , Biofilmes , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Fosforilação , Regiões Promotoras Genéticas , Conformação Proteica , Transdução de Sinais , Vibrio cholerae/genéticaRESUMO
Protein cages have recently emerged as an extraordinary drug-delivery system due to its biocompatibility, biodegradability, low toxicity, ease to manipulate and engineer. We have reported earlier the formation and architecture of a do-decameric cage-like architecture of Vibrio cholerae acylphosphatase (VcAcP) at 3.1 Å. High resolution (2.4 Å) crystal structure of VcAcP cage, reported here, illuminates a potential binding site for sulphate/phosphate containing drugs whereas analysis of its subunit association and interfaces indicates high potential for cage engineering. Tryptophan quenching studies indeed discloses noteworthy binding with various sulphate/phosphate containing nucleotide-based drugs and vitamin B6 (PLP) demonstrating that exterior surface of VcAcP protein cage can be exploited as multifunctional carrier. Moreover, a quadruple mutant L30C/T68C/N40C/L81C-VcAcP (QM-VcAcP) capable to form an intricate disulphide bonded VcAcP cage has been designed. SEC, SDS-PAGE analysis and DLS experiment confirmed cysteine mediated engineered VcAcP cage formation.
Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vibrio cholerae/enzimologia , Hidrolases Anidrido Ácido/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cromatografia em Gel , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Difusão Dinâmica da Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Vibrio cholerae/genética , AcilfosfataseRESUMO
Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3'3'-cyclic GMP-AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5'-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5'-pGpG-Ca2+ structure, ß5-α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5'-pGpG-Ca2+ structure quite different from other 5'-pGpG bound structures reported earlier.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Nucleotídeos Cíclicos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/genética , Biofilmes , GMP Cíclico/química , GMP Cíclico/metabolismo , Nucleotídeos Cíclicos/química , Diester Fosfórico Hidrolases/genética , Vibrio cholerae/química , Vibrio cholerae/genética , Vibrio cholerae/fisiologiaRESUMO
Rho-dependent transcription termination is a well-conserved process in bacteria. The Psu and YaeO proteins are the two established inhibitors of the ATP-dependent RNA helicase Rho protein of Escherichia coli. Here, we show a detailed sequence and phylogenetic analysis demonstrating that Vibrio cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO induces significant growth defect on in vivo expression and inhibits in vitro functions of the V. cholerae Rho on directly binding to the latter. Through various biophysical techniques, we showed that interaction of VcYaeO disrupts the oligomeric state of the VcRho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR structure of the EcYaeO. Interestingly, VcYaeO structurally resembles the Hfq protein, and like the latter, it exhibits ssDNA/RNA-binding properties. Docking studies demonstrate probable interactions of VcYaeO with VcRho and mode of inhibition of RNA binding to Rho. We propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter's hexameric assembly and also likely by sequestering the RNA from the Rho primarybinding sites.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Fator Proteico 1 do Hospedeiro/química , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fator Rho/química , Fator Rho/isolamento & purificação , Análise de Sequência de DNA , Relação Estrutura-AtividadeRESUMO
Low molecular weight protein tyrosine phosphatases (LMWPTPs) are ubiquitously found as small cytoplasmic enzymes which act on phospho-tyrosine containing proteins that are engaged in various cellular functions. Vibrio cholerae O395 contains two LMWPTPs having widely different sequence. Phylogenetic analysis based on a non redundant set of 124 LMWPTP sequences, designate that LMWPTP-2 from Vibrio choleraeO395 (VcLMWPTP-2) is a single taxon. We have determined the crystal structure of VcLMWPTP-2 at 2.6â¯Å with MOPS bound in the active site. Tertiary structure analysis indicates that VcLMWPTP-2 forms dimer. Studies in solution state also confirm exclusive presence of a dimeric form. Kinetic studies demonstrate that VcLMWPTP-2 dimer is catalytically active while inactivation through oligomerisation was reported as one of the regulatory mechanism in case of mammalian LMWPTP viz., Bos taurus LMWPTP, BPTP. Kinetic studies using p-nitrophenyl phosphate (p-NPP) as a substrate demonstrate active participation of both the P-loop cysteine in catalysis. Vicinal Cys17, in addition plays a role of protecting the catalytic Cys12 under oxidative stress. Structural analysis and MD simulations allowed us to propose the role of several conserved residues around the active site. Distribution of surface charges and grooves around the active site delineates unique features of VcLMWPTP-2 which could be utilized to design specific inhibitor.
Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Catálise , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Cinética , Proteínas de Membrana/química , Modelos Moleculares , Peso Molecular , Filogenia , Ligação Proteica/fisiologia , Proteínas Tirosina Fosfatases/ultraestrutura , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/fisiologia , Vibrio cholerae/química , Vibrio cholerae/genéticaRESUMO
Fructokinase (FRK) catalyzes the first step of fructose metabolism i.e., D-fructose to D-fructose-6-phosphate (F6P), however, the mechanistic insights of this reaction are elusive yet. Here we demonstrate that the putative Vibrio cholerae fructokinase (VcFRK) exhibit strong fructose-6-kinase activity allosterically modulated by K+/Cs+. We have determined the crystal structures of apo-VcFRK and its complex with fructose, fructose-ADP-Ca2+, fructose-ADP-Ca2+-BeF3-. Collectively, we propose the catalytic mechanism and allosteric activation of VcFRK in atomistic details explaining why K+/Cs+ are better activator than Na+. Structural results suggest that apo VcFRK allows entry of fructose in the active site, sequester it through several conserved H-bonds and attains a closed form through large scale conformational changes. A double mutant (H108C/T261C-VcFRK), that arrests the closed form but unable to reopen for F6P release, is catalytically impotent highlighting the essentiality of this conformational change. Negative charge accumulation around ATP upon fructose binding, is presumed to redirect the γ-phosphate towards fructose for efficient phosphotransfer. Reduced phosphotransfer rate of the mutants E205Q and E110Q supports this view. Atomic resolution structure of VcFRK-fructose-ADP-Ca2+-BeF3-, reported first time for any sugar kinase, suggests that BeF3- moiety alongwith R176, Ca2+ and 'anion hole' limit the conformational space for γ-phosphate favoring in-line phospho-transfer.
Assuntos
Frutoquinases/química , Frutoquinases/metabolismo , Modelos Moleculares , Conformação Molecular , Açúcares/química , Açúcares/metabolismo , Vibrio cholerae/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
Vibrio cholerae, experiences a highly hostile environment at human intestine which trigger the induction of various heat shock genes. VcHsp15, the hslR gene product of V. cholerae O395 is a highly up regulated protein which targets erroneously dislodged 50S subunit upon heat shock that carries a tRNA attached to the abortive nascent polypeptide chain, and recycle it for another round of translation. In this study we report the crystal structure of VcHsp15â¯at 2.33â¯Å. Although the structure of VcHsp15 share very similar fold to E. Coli Hsp15 their oligomerization properties are quite different. While EcHsp15 is a monomer, VcHsp15 exhibit a novel trimeric form both in crystal structure and in solution. The putative αL motif of VcHsp15 shares a strikingly similar fold with several RNA binding proteins like ribosomal protein S4 and threonyl-tRNA synthetase. Curiously, their αL motif display a comparable surface charge, albeit extremely low sequence identity, indicating that this motif serves as a basic module to bind RNA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Proteínas de Choque Térmico/química , Multimerização Proteica , Proteínas de Ligação a RNA/química , Vibrio cholerae/química , Cristalografia por Raios X , HumanosRESUMO
Vibrio cholerae experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The hchA gene product of V. cholerae O395, referred to a hypothetical intracellular protease/amidase VcHsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that VcHsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on VcHsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. VcHsp31 suppresses the aggregation of insulin in vitro in a dose dependent manner. Through crystal structures of VcHsp31 and its mutants, grown at various temperatures, we demonstrate that VcHsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to EcHsp31 where two VcHsp31 monomers associate in eclipsed manner through several intersubunit hydrogen bonds involving their P-domains. Type-II dimer is a novel dimeric organization, where some of the intersubunit hydrogen bonds are abrogated and each monomer swings out in the opposite directions centering at their P-domains, like twisting of wet cloth. Normal mode analysis (NMA) of Type-I dimer shows similar movement of the individual monomers. Upon swinging, a dimeric surface of ~400Å2, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, VcHsp31 remains as an equilibrium mixture of both the dimers. With increase in temperature, transformation to Type-II form having more exposed hydrophobic surface, occurs progressively accounting for the temperature dependent increase of chaperone activity of VcHsp31.
Assuntos
Proteínas de Bactérias/química , Proteínas de Choque Térmico/química , Lactoilglutationa Liase/química , Peptídeo Hidrolases/química , Vibrio cholerae/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Insulina/química , Insulina/metabolismo , Cinética , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Vibrio cholerae/enzimologiaRESUMO
The crystal structure of phosphoglucomutase (LmPGM) from the parasite Leishmania major has been solved at 3.5 Å resolution. Although the active form of the enzyme is monomeric in solution, four molecules (A, B, C, D) were found in the asymmetric unit, of which the pairs (A,D) and (B,C) were of identical inter-subunit geometry. The parasitic enzyme constituted of four domains exhibited the canonical 'heart' shape of the protein, with domains I to III having a conserved α|ß core, while the fourth (IV) domain being structurally distinct from the rest. Conformational variability of the IVth domain, postulated to be responsible for the catalytic function of the enzyme has been studied by normal mode analysis (NMA) and the conformational features responsible for domain movement in the 'hinge region' analyzed in detail. Although the active site of phosphoglucomutase is highly conserved from parasite to human, initial calculations show that a ligand binding pocket could exist near the hinge region, which is unique to the parasite. The enzymatic parameters of LmPGM have been determined and compared with other PGMs from orthologous species in addition to elucidating its mechanism of action by docking the substrate, intermediate onto the active site.
Assuntos
Leishmania major/enzimologia , Modelos Moleculares , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Conformação Proteica , Especificidade por SubstratoRESUMO
Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
ß-trefoil fold, consisting of a six stranded ß-barrel capped at one end by a lid comprising of another six ß-strands, is one of the most important folds among proteins. Important classes of proteins like Interleukins (ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Their core is packed by hydrophobic residues contributed by the 6 stranded ß-barrel and three ß-hairpins that make essential contacts with each other and keep the protein in 'topologically minimal frustrated state'. A complete database analysis of the core residues of the ß-trefoil fold proteins presented here identified a conserved tryptophan (W91) residue in the Kunitz (STI) family of inhibitors that projects from the lid and interacts with the bottom layer residues of the barrel. This kind of interactions is unique in Kunitz (STI) family because no other families of ß-trefoil fold have such a shear sized residue at the barrel lid junction; suggesting its possible importance in packing and stability. We took WCI as a representative of this family and prepared four cavity creating mutants W91F-WCI, W91M-WCI, W91I-WCI & W91A-WCI. CD experiments show that the secondary structure of the mutants remains indistinguishable with the wild type. Crystal structures of the mutants W91F-WCI, W91M-WCI & W91A-WCI also show the same feature. However, slight readjustments of the side chains around the site of mutation have been observed so as to minimize the cavity created due to mutation. Comparative stability of these mutants, estimated using heat denaturation CD spectroscopy, indicates that stability of the mutants inversely correlates with the size of the cavity inside the core. Interestingly, although we mutated at the core, mutants show varying susceptibility against tryptic digestion that grossly follow their instability determined by CD. Our findings suggest that the W91 residue plays an important role in determining the stability and packing of the core of WCI.
Assuntos
Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Inibidor da Tripsina de Soja de Kunitz/química , Triptofano/química , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Interleucinas/química , Interleucinas/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Estabilidade Proteica , Temperatura , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Triptofano/genética , Triptofano/metabolismoAssuntos
Proteínas de Bactérias/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Multimerização Proteica , Estrutura Secundária de Proteína , Vibrio cholerae/enzimologia , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos/química , Domínio Catalítico , Cátions Monovalentes/química , Cátions Monovalentes/metabolismo , Cátions Monovalentes/farmacologia , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Ribose/química , Ribose/metabolismo , Homologia de Sequência de Aminoácidos , Vibrio cholerae/genéticaRESUMO
Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, the rbsK gene from Vibrio cholerae O395 encoding ribokinase was cloned and the protein was overexpressed in Escherichia coli BL21 (DE3) and purified using Ni(2+)-NTA affinity chromatography. Crystals of V. cholerae RK (Vc-RK) and of its complex with ribose and ADP were grown in the presence of polyethylene glycol 6000 and diffracted to 3.4 and 1.75â Å resolution, respectively. Analysis of the diffraction data showed that both crystals possess symmetry consistent with space group P1. In the Vc-RK crystals, 16 molecules in the asymmetric unit were arranged in a spiral fashion, leaving a large empty space inside the crystal, which is consistent with its high Matthews coefficient (3.9â Å(3)â Da(-1)) and solvent content (68%). In the Vc-RK co-crystals four molecules were located in the asymmetric unit with a Matthews coefficient of 2.4â Å(3)â Da(-1), corresponding to a solvent content of 50%.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Vibrio cholerae/enzimologia , Sequência de Bases , Cristalização , Cristalografia por Raios X , Primers do DNA , Reação em Cadeia da PolimeraseRESUMO
Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization in a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.
Assuntos
Modelos Químicos , Modelos Moleculares , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/ultraestrutura , Vibrio cholerae/enzimologia , Sítios de Ligação , Catálise , Simulação por Computador , Dimerização , Peso Molecular , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases/classificaçãoRESUMO
Here we show the formation of an ~8-nm cage formed by the self-assembly of acylphosphatase from Vibrio cholerae O395 (Vc-AcP). The 12-subunit cage structure forms spontaneously and is stabilized through binding of sulfate ions at its exterior face and interfacial regions. Crystal structure and studies in solutions illuminate the basis for the formation of the cage, while a single (Cys20âArg) mutation (Vc-AcP-C20R) transforms Vc-AcP to a potent enzyme but disrupts the assembly into a trimer.
Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Multimerização Proteica , Vibrio cholerae/enzimologia , Substituição de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação Proteica , AcilfosfataseRESUMO
Vibrio cholerae contains multiple copies of chemotaxis response regulator (VcCheY1-VcCheY4) whose functions are elusive yet. Although previous studies suggested that only VcCheY3 directly switches the flagellar rotation, the involvement of VcCheY4 in chemotaxis could not be ruled out. None of these studies, however, focused on the structure, mechanism of activation or molecular basis of FliM binding of the VcCheYs. From the crystal structures of Ca(2+) and Mg(2+) bound VcCheY3 we proposed the presence of a conformational barrier composed of the hydrophobic packing of W61, M88 and V106 and a unique hydrogen bond between T90 and Q97 in VcCheY3. Lesser fluorescence quenching and higher Km value of VcCheY3, compared to its mutants VcCheY3-Q97A and VcCheY3-Q97A/E100A supported our proposition. Furthermore, aforesaid biochemical data, in conjunction with the structure of VcCheY3-Q97A, indicated that the coupling of T90 and Q97 restricts the movement of T90 toward the active site reducing the stabilization of the bound phosphate and effectively promoting autodephosphorylation of VcCheY3. The structure of BeF3(-) activated VcCheY3 insisted us to argue that elevated temperature and/or adequacy of phosphate pool might break the barrier of the free-state VcCheY3 and the conformational changes, required for FliM binding, occur upon phosphorylation. Structure of VcCheY4 has been solved in the free and sulfated states. VcCheY4(sulf), containing a bound sulfate at the active site, appears to be more compact and stable with a longer α4 helix, shorter ß4α4 loop and hydrogen bond between T82 and the sulfate compared to VcCheY4(free). While pull down assay of VcCheYs with VcFliMNM showed that only activated VcCheY3 can interact with VcFliMNM and VcCheY4 cannot, a knowledge based docking explained the molecular mechanism of the interactions between VcCheY3 and VcFliM and identified the limitations of VcCheY4 to interact with VcFliM even in its phosphorylated state.
Assuntos
Proteínas de Bactérias/metabolismo , Vibrio cholerae/metabolismo , Ligação Proteica/fisiologia , Conformação ProteicaRESUMO
The conserved bacterial transcription terminator, Rho, is a potent target for bactericidal agents. Psu, a bacteriophage P4 capsid protein, is capable of inducing anti-termination to the Rho-dependent transcription termination. Knowledge of structural and mechanistic basis of this anti-termination is required to design peptide-inhibitor(s) of Rho from Psu. Using suppressor genetics, cross-linking, protein foot-printing and FRET analyses, we describe a conserved disordered structure, encompassing 139-153 amino acids of Rho, as the primary docking site for Psu. Also a neighbouring helical structure, comprising 347-354 amino acids, lining its central channel, plays a supportive role in the Rho-Psu complex formation. Based on the crystal structure of Psu, its conformation in the capsid of the P4 phage, and its interacting regions on Rho, we build an energy-minimized structural model of the Rho:Psu complex. In this model, a V-shaped dimer of Psu interacts with the two diagonally opposite subunits of a hexameric Rho, enabling Psu to form a 'lid' on the central channel of the latter. We show that this configuration of Psu makes the central channel of Rho inaccessible, and it causes a mechanical impediment to its translocase activity.
Assuntos
Proteínas do Capsídeo/química , Fator Rho/química , Terminação da Transcrição Genética , Adenosina Trifosfatases/antagonistas & inibidores , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/genética , Reagentes de Ligações Cruzadas , Cisteína/química , Ácido Edético/análogos & derivados , Ácido Edético/química , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Mutação , Pegadas de Proteínas , Fator Rho/genética , Fator Rho/metabolismoRESUMO
Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg(2+) single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfide-bridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.
Assuntos
Bacteriófagos/metabolismo , Proteínas do Capsídeo/química , Myoviridae/metabolismo , Sequência de Aminoácidos , Bacteriófagos/química , Bacteriófagos/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Dimerização , Enterobacteriaceae/virologia , Modelos Moleculares , Dados de Sequência Molecular , Myoviridae/química , Myoviridae/genética , Dobramento de Proteína , Alinhamento de SequênciaRESUMO
Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75â Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P2(1)2(1)2, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4â Å3â Da(-1), which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1â Å3â Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.