Susmita Khamrui

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed “mini‐proteins,” containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop‐scaffold compatibility, we prepared three chimeric proteins ECIL‐WCIS, ETIL‐WCIS, and STIL‐WCIS, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECIL‐WCIS and STIL‐WCIS behave like good inhibitors, ETIL‐WCIS behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETIL‐WCIS shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL‐WCIS leading to a loss of canonical conformation, explaining its substrate‐like behavior. Incorporation of this barrier back in ETIL‐WCIS through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop‐scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.

The first structure of Polarity Suppression protein, Psu from Enterobacteria phage P4, reveals a novel fold and a knotted dimer

Background: Phage P4 Psu protein is a capsid decoration protein with unknown structure. Results: The first structure of Psu reveals a novel fold and a knotted dimer. Conclusion: The V-shaped molecular architecture is important for capsid binding. Significance: The structure of Psu will help to design peptide fragments, which can be used as drugs against the bacterial transcription machinery. 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 Hg2+ 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.

Conformational Barrier of CheY3 and Inability of CheY4 to Bind FliM Control the Flagellar Motor Action in Vibrio cholerae

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 Ca2+ and Mg2+ 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. VcCheY4sulf, containing a bound sulfate at the active site, appears to be more compact and stable with a longer α4 helix, shorter β4α4 loop and hydrogen bond between T82 and the sulfate compared to VcCheY4free. 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.

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.

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, β = 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°.

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.