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.

Fig 1. Cartoon representation and sequence alignment of VcHsp31 with other orthologs showing domain organisation and catalytic residues.

(a) Cartoon representation of the overall structure of VcHsp31 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) 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 VcHsp31 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.

Fig 2. Superpositions of VcHsp31 with three different Hsp31 orthologs.

(a) EcHsp31 (Class-I) [PDB ID 1PV2, sequence identity 58.885% (calculated in ClustalO), rmsd 0.428] [15] (b) YDR533Cp from Sachharomyces cerevisiae (Class-II) [PDB ID 4QYX, sequence identity 18.033%, rmsd 2.932] [41] and (c) PH1704 from Pyrococcus horikoshii (Class-III) [PDB ID 1G2I, sequence identity 10.69%, rmsd 1.533] [40] Hsps to show their domain organization. VcHsp31 is shown in same color as in Fig 1a).

Fig 3. Cartoon representation of VcHsp31 dimer in different orientations and their electrostatic surface (a-b) looking down the twofold symmetry and (c) perpendicular the twofold.

Electron density contoured at 1σ, overlaid on the linker region (in sticks), is shown in blue mesh (Top). Electrostatic potential surface of VcHsp31 dimer shown with an orientation same as in cartoon representation. Canyon (yellow dots), bowl and the location of the catalytic triad (green arrow) are indicated (bottom). Color code used is same as Fig 1a, a lighter shade is used for the other monomer.

Fig 4. Interactions at the dimeric interface in Type-I and Type-II dimers.

(a) Interactions at the A-B and C-D dimeric interface of VcHsp31. 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).

Fig 5. Type-I and Type-II dimer of VcHsp31.

(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 VcHsp31at low temperatures (18°-25°C).

Fig 6. B-factor plot of different dimers.

(a) Temperature factor plot of Type-I dimers of VcHsp31^20C, B averages of chain A (red) and chain E (green) are plotted as representative (b) B averages of Type-II dimers of VcHsp31^25C 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.

Fig 7. Access of catalytic triad through two cavity pocket.

(a) Residues involved in forming the two cavity pocket in EcHsp31. Cavity 1 and Cavity 2 are indicated by arrow; (b) Two cavity pocket in VcHsp31 in the same orientation to that of EcHsp31; (c) Superposition of residues forming the two cavity pocket in EcHsp31 and VcHsp31. Residues of VcHsp31 that differ in sequence or having structural alterations with EcHsp31 are only labeled; (d) Temperature dependence of amidopeptidase activity of VcHsp31 determined using Ala-AMC as substrate.

Fig 8. Influence of highly conserved residues around catalytic site on peptidase and methylglyoxalase activity.

(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 VcHsp31 plotted against substrate concentration. (d) Methylglyoxalase activity of different VcHsp31 mutants.

Fig 9. VcHsp31 inhibits in vitro protein aggregation.

DTT induced aggregation of insulin and suppression by VcHsp31 at 35°C. Insulin (stock concentration 3 mg/ml in 20 mM Tris buffer, pH-8.0, containing 150 mM NaCl dissolved in presence of 20 mM NaOH) was reduced with 25 mM DTT in a final volume of 1.2 ml and aggregation of insulin in the absence of VcHsp31(black trace) and presence of VcHsp31 with insulin in the substrate:enzyme ratio of 15:1 (red trace), 3:1 (blue trace), 1:1 (green trace) and 1:6 (violet trace) was monitored by measuring the right angle light scattering at 360 nm. The graph is a representative plot (plotted in originpro 8) of three individual experiments.