title: Structural and biochemical studies on Vibrio cholerae Hsp31 reveals a novel dimeric form and Glutathione-independent Glyoxalase activity.
abstract: 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.
authors.affiliation: Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India.
authors.forename: Samir
authors.initials: S
authors.lastname: Das
authors.affiliation: Crystallography and Molecular Biology Division Saha Institute of Nuclear Physics, Kolkata, India.
authors.forename: Sanghati
authors.initials: S
authors.lastname: Roy Chowdhury
authors.affiliation: Department of Biotechnology, St. Xavier's College, Kolkata.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Dey
authors.affiliation: Crystallography and Molecular Biology Division Saha Institute of Nuclear Physics, Kolkata, India.
authors.forename: Udayaditya
authors.identifier: http://orcid.org/0000-0001-5742-2907
authors.initials: U
authors.lastname: Sen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005978:Glutathione; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D007328:Insulin; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D007791:Lactoylglutathione Lyase; D005978:Glutathione