Cartoon representation and sequence alignment of <i>Vc</i>Hsp31 with other orthologs showing domain organisation and catalytic residues.

<p><b>(a)</b> Cartoon representation of the overall structure of <i>Vc</i>Hsp31 monomer where ‘A’ domain (green), ‘P’ domain (violet) and the ‘linker’ region (yellow) are indicated. The catalytic triad region comprising Cys188-His189-Asp216 (small red box) is zoomed for clarity; <b>(b)</b> Structure based sequence alignment of the three classes of Hsps taking two representative members from each class. Top of the alignment depicts their secondary structures and every twentieth residue of <i>Vc</i>Hsp31 is marked by a (|). At the bottom of the alignments, P domain segments and ‘linker region’ are indicated by colored bars. Catalytic residues are marked in red dots while residues conserved in all six proteins are in black dots.</p

Access of catalytic triad through two cavity pocket.

<p>(a) Residues involved in forming the two cavity pocket in <i>Ec</i>Hsp31. Cavity 1 and Cavity 2 are indicated by arrow; (b) Two cavity pocket in <i>Vc</i>Hsp31 in the same orientation to that of <i>Ec</i>Hsp31; (c) Superposition of residues forming the two cavity pocket in <i>Ec</i>Hsp31 and <i>Vc</i>Hsp31. Residues of <i>Vc</i>Hsp31 that differ in sequence or having structural alterations with <i>Ec</i>Hsp31 are only labeled; (d) Temperature dependence of amidopeptidase activity of <i>Vc</i>Hsp31 determined using Ala-AMC as substrate.</p

Interactions at the dimeric interface in Type-I and Type-II dimers.

<p>(a) Interactions at the A-B and C-D dimeric interface of <i>Vc</i>Hsp31. Two monomers are shown in yellow and brown and strong electrostatic interactions are shown in dashed line. (b) Electron density contoured at 1σ at the E-F dimeric interface showing that the salt bridge between K105 and E60 is abolished here (red label).</p

Type-I and Type-II dimer of <i>Vc</i>Hsp31.

<p>(a) Overall superposition of the dimers (viewing perpendicular to the bowl). Direction of swinging motion required to form Type-II dimer from Type-I dimer is shown by the flat arrow and the position of pivot point is shown in red triangle; (b) Disposition of ‘chain B’ and ‘chain F’ (viewing from top of the canyon) when ‘chain A’ and ‘chain E’ are superposed. Large displacements of helices are evident here; (c) Same superposition scheme as in Fig 5b but chain A is shown as surface (viewing same as Fig 5a) which shows poor packing of α4 for Type-II dimer with chain E (shown in surface) (d) A portion of buried dimeric surface in Type-I dimer which is being exposed (e) in Type-II dimers; (f) Tryptophan quenching of <i>Vc</i>Hsp31at low temperatures (18°-25°C).</p

Structural and biochemical studies on <i>Vibrio cholerae</i> Hsp31 reveals a novel dimeric form and Glutathione-independent Glyoxalase activity

<div><p><i>Vibrio cholerae</i> experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The <i>hchA</i> gene product of <i>V</i>. <i>cholerae</i> O395, referred to a hypothetical intracellular protease/amidase <i>Vc</i>Hsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that <i>Vc</i>Hsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on <i>Vc</i>Hsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. <i>Vc</i>Hsp31 suppresses the aggregation of insulin <i>in vitro</i> in a dose dependent manner. Through crystal structures of <i>Vc</i>Hsp31 and its mutants, grown at various temperatures, we demonstrate that <i>Vc</i>Hsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to <i>Ec</i>Hsp31 where two <i>Vc</i>Hsp31 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 ~400Ų, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, <i>Vc</i>Hsp31 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 <i>Vc</i>Hsp31.</p></div

Influence of highly conserved residues around catalytic site on peptidase and methylglyoxalase activity.

<p>(a) Amino acid residues around the catalytic C188 of VcHsp31 which are mutated for functional studies. Among them residues in ball-and-stick are those for which functional studies have been done. Residues in stick were mutated but the resulting protein has poor solubility and eventually not included for functional studies. Residues Met225 and Phe71 are shown in dot-surface. (b) Peptidase activity of different mutants measured using Ala-AMC as substrate at 37°C. (c) Methylglyoxalase activity of <i>Vc</i>Hsp31 plotted against substrate concentration. (d) Methylglyoxalase activity of different <i>Vc</i>Hsp31 mutants.</p

Average B-factors of different chains of <i>Vc</i>Hsp31.

<p>Average B-factors of different chains of <i>Vc</i>Hsp31.</p

Interface area (Ų) and dimer types for different <i>Vc</i>Hsp31 crystals.

<p>Interface area (Ų) and dimer types for different <i>Vc</i>Hsp31 crystals.</p

B-factor plot of different dimers.

<p>(a) Temperature factor plot of Type-I dimers of <i>Vc</i>Hsp31^20C, B averages of chain A (red) and chain E (green) are plotted as representative (b) B averages of Type-II dimers of <i>Vc</i>Hsp31^25C are plotted with chains A (red), C (blue), E (green) as representative. Regions α4/β7/α5 and β4/β5 loop, that loose contact at the dimeric surface upon swinging motion in type-II dimer are shown by asterisk.</p

Interactions at the interface of A-B and E-F dimer.

<p>Interactions at the interface of A-B and E-F dimer.</p