High resolution structure of Vibrio cholerae acylphosphatase (VcAcP) cage: Identification of drugs, location of its binding site and engineering to facilitate cage formation.

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.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication.

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.

Crystal structure of heat shock protein 15 (Hsp15) from Vibrio cholerae: Novel mode of trimerization and nucleic acid binding properties.

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 VcHsp15 at 2.33 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.

Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.

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.

A conserved tryptophan (W91) at the barrel-lid junction modulates the packing and stability of Kunitz (STI) family of inhibitors.

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.

Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.

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.

Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family.

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.

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications.

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.

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.

Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors.

Canonical serine protease inhibitors interact with cognate enzymes through the P3-P2' region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETI(L)-WCI(S), having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETI(L)-WCI(S)), where the barrier is reintroduced on ETI(L)-WCI(S), shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETI(L)-WCI(S)) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.

Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain.

APP intracellular C-terminal domain (AICD-47), generated upon γ-secretase cleavage of amyloid precursor's protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine-binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the "conformational switching" of AICD-47 becomes evident. In order to understand different binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spectroscopy has revealed an overall random coil like structure in different pHs while TFE (2'-2'-2'-trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced α-helicity to a certain extent. Binding of Tyr phosphorylated AICD-47 ((P)AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (ΔH=-197.5±6.2 kcal mole(-1) at 25 °C) and an unfavorable entropic contribution (ΔS=-631 cal mol(-1) deg(-1) at 25 °C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer's disease phenotype.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of a fructokinase from Vibrio cholerae O395.

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 1.75 Šresolution, 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 2.4 Å3 Da(-1), which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1 Å3 Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.

Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae.

Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni(2+)-NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P3(1) and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å(3) Da(-1) and 55.62%, respectively.

Crystal Structure of VpsR Revealed Novel Dimeric Architecture and c-di-GMP Binding Site: Mechanistic Implications in Oligomerization, ATPase Activity and DNA Binding.

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.

Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395.

Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1-CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, ß = 96.5°, whereas crystals grown in space group P3(2)21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDa Vibrio cholerae heat-shock protein VcHsp31.

The Gram-negative bacterium Vibrio cholerae, which is responsible for the diarrhoeal disease cholera in humans, induces the expression of numerous heat-shock genes. VcHsp31 is a 31 kDa putative heat-shock protein that belongs to the DJ-1/PfpI superfamily, functioning as both a chaperone and a protease. VcHsp31 has been cloned, overexpressed and purified by Ni(2+)-NTA affinity chromatography followed by gel filtration. Crystals of VcHsp31 were grown in the presence of PEG 6000 and MPD; they belonged to space group P2(1) and diffracted to 1.9 Å resolution. Assuming the presence of six molecules in the asymmetric unit, the Matthews coefficient was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%.

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae.

Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.

Crystallization and preliminary X-ray analysis of Psu, an inhibitor of the bacterial transcription terminator Rho.

Psu, a coat protein from bacteriophage P4, inhibits Rho-dependent transcription termination both in vivo and in vitro. The Psu protein is alpha-helical in nature and appeared to be a dimer in solution. It interacts with Rho and affects the ATP binding and RNA-dependent ATPase activity of Rho, which in turn reduces the rate of RNA release from the elongation complex. Crystals of Psu were grown in space group I422 in the presence of PEG, with unit-cell parameters a = b = 148.76, c = 63.38 A and a calculated Matthews coefficient of 2.1 A(3) Da(-1) (41.5% solvent content), assuming the presence of two molecules in the asymmetric unit. A native data set was collected to 2.3 A resolution.

Crystal structure of the human lymphoid tyrosine phosphatase catalytic domain: insights into redox regulation .

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, recently emerged as an important risk factor and drug target for human autoimmunity. Here we solved the structure of the catalytic domain of LYP, which revealed noticeable differences with previously published structures. The active center with a semi-closed conformation binds a phosphate ion, which may represent an intermediate conformation after dephosphorylation of the substrate but before release of the phosphate product. The structure also revealed an unusual disulfide bond formed between the catalytic Cys and one of the two Cys residues nearby, which is not observed in previously determined structures. Our structural and mutagenesis data suggest that the disulfide bond may play a role in protecting the enzyme from irreversible oxidation. Surprisingly, we found that the two noncatalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structural and functional characterizations have provided new insights into autoregulatory mechanisms of LYP function.