The 1.6-A crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad

Abstract

Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-A crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The alpha-beta sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.

Figure 1

Ribbon representation of the EcHsp31 monomer. The A domain is blue, the P domain is green (P1–P3 segments are in progressively lighter shades of green), and the linker region is purple. The catalytic triad is shown as a red ball-and-stick model (28).

Figure 2

Putative active site pocket of EcHsp31. (A) Surface representation of the active site pocket. Surfaces from the A and P domains are blue and green, respectively. Two residues of the triad, Cys-185 in yellow and His-186 in red, are visible. (B) Residues contributing to the active site pocket. Residues surrounding the pocket are blue (A domain) and green (P domain). The catalytic triad is colored by atom type: carbons are green, oxygens are red, nitrogens are blue, and sulfurs are yellow. Cavity 1 residues are red and cavity 2 residues are orange (29).

Figure 3

Two views of ribbon representation of the EcHsp31 dimer. The A and P domains of subunit I are blue and green, respectively, and are pale blue and pale green for subunit II, respectively. The linker is purple and light purple for subunits I and II, respectively. The catalytic triads of both subunits are shown in the red ball-and-stick illustration (28). (A) View down the crystallographic twofold axis. (B) View perpendicular to the crystallographic twofold axis.

Figure 4

Two views of the electrostatic potential of the EcHsp31 dimer. Each view was generated with grasp (26). Electrostatic surfaces are colored between −10 kT (red) and +10 kT (blue). Canyon and bowl are labeled. The negative groove connecting the putative triads is shown by a black line, with arrows pointing to triad(s). (A) View down the crystallographic twofold axis. (B) View perpendicular to the twofold axis, looking through the canyon of the A domains.

Figure 5

Structural comparison of EcHsp31 and PhPI. The A and P domains of EcHsp31 are blue and green, respectively, and PhPI is orange. The catalytic triad of EcHsp31 is colored by atom type: carbons are green, oxygens are red, nitrogens are blue, and sulfurs are yellow. (A) Schematic topology diagram of EcHsp31 and PhPI (30). Triangles represent β-strands, large circles represent α-helices, and small circles represent 3₁₀ helices. Secondary structure is named as in EcHsp31 and PhPI models. (B) Stereoview of the superposition of the C^α ribbon trace of EcHsp31 and PhPI (29). (C) View of the catalytic triads of EcHsp31 and PhPI. Subunits A and C of PhPI are orange and yellow, respectively. The PhPI triad residues for Cys-100 and His-185 are orange, and Glu-74′ is yellow (29).

Figure 6

Structure-guided sequence alignment of the Hsp31 family of proteins. Class I, class II, and class III members of the Hsp31 family are designated with blue, black, and orange roman numerals, respectively. Red boxes show 100% conservation across the Hsp31 family. The secondary structure for EcHsp31 is shown above the sequence and is colored by domain, whereas the secondary structure for PhPI is shown below the sequence in orange. The catalytic triad of EcHsp31 (Cys-185, His-186, and Asp-214) is marked with red stars and the catalytic triad of PhPI (Cys-100, His-101, and Glu-74′) is marked with orange stars. The third member (Asp-214 or Glu-74′) of the triad has a large star. Purple boxes show residues corresponding to Asp-214 in class I and II. Blue boxes in class I indicate dimer interactions of EcHsp31. Yellow (A–C interaction) and orange (A–B interaction) boxes in class III indicate hexamer interactions of PhPI (31).