Intrinsic Disorder and Other Malleable Arsenals of Evolved Protein Multifunctionality.

Microscopic evolution at the functional biomolecular level is an ongoing process. Leveraging functional and high-throughput assays, along with computational data mining, has led to a remarkable expansion of our understanding of multifunctional protein (and gene) families over the past few decades. Various molecular and intermolecular mechanisms are now known that collectively meet the cumulative multifunctional demands in higher organisms along an evolutionary path. This multitasking ability is attributed to a certain degree of intrinsic or adapted flexibility at the structure-function level. Evolutionary diversification of structure-function relationships in proteins highlights the functional importance of intrinsically disordered proteins/regions (IDPs/IDRs) which are highly dynamic biological soft matter. Multifunctionality is favorably supported by the fluid-like shapes of IDPs/IDRs, enabling them to undergo disorder-to-order transitions upon binding to different molecular partners. Other new malleable members of the protein superfamily, such as those involved in fold-switching, also undergo structural transitions. This new insight diverges from all traditional notions of functional singularity in enzyme classes and emphasizes a far more complex, multi-layered diversification of protein functionality. However, a thorough review in this line, focusing on flexibility and function-driven structural transitions related to evolved multifunctionality in proteins, is currently missing. This review attempts to address this gap while broadening the scope of multifunctionality beyond single protein sequences. It argues that protein intrinsic disorder is likely the most striking mechanism for expressing multifunctionality in proteins. A phenomenological analogy has also been drawn to illustrate the increasingly complex nature of modern digital life, driven by the need for multitasking, particularly involving media.

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.

Vibrio cholerae LMWPTP-2 display unique surface charge and grooves around the active site: Indicative of distinctive substrate specificity and scope to design specific inhibitor.

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 2.6 Šwith 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.

Large-scale conformational changes and redistribution of surface negative charge upon sugar binding dictate the fidelity of phosphorylation in Vibrio cholerae fructokinase.

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.

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.

A novel 8-nm protein cage formed by Vibrio cholerae acylphosphatase.

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 (Cys20→Arg) mutation (Vc-AcP-C20R) transforms Vc-AcP to a potent enzyme but disrupts the assembly into a trimer.

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.

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.