The linker-loop region ofEscherichia colichaperone Hsp31 functions as a gate that modulates high-affinity substrate binding at elevated temperatures

Abstract

Precise control of substrate binding and release is essential for molecular chaperones to exert their protective function in times of stress. The mechanisms used are diverse and have been difficult to unravel. Escherichia coli heat-shock protein 31 (Hsp31) is a recent addition to the known complement of eubacterial chaperones. Crystallographic studies have revealed the presence of a hydrophobic bowl at the Hsp31 dimer interface and shown that the linker region connecting the two structural domains within each subunit is disordered. Together with a neighboring flexible loop, the linker caps a hydrophobic area adjacent to the bowl. Using a collection of Hsp31 mutants, we show that although both bowl and linker-loopshielded residues participate in substrate binding, the latter are critical for protein capture at high temperature. Linker immobilization via an artificial disulfide bridge abolishes chaperone activity at elevated temperatures by precluding exposure of the underlying hydrophobic domain. We conclude that Hsp31 uses its linker-loop region as a thermally activated gate to control nonnative protein annealing to a high-affinity substrate-binding site. This simple yet efficient strategy to capture partially folded proteins under heatshock conditions may be shared by other folding modulators. molecular chaperones ͉ heat-shock proteins ͉ structure function ͉ YedU ͉ HchA

Integrity of N‐ and C‐termini is important for E. coli Hsp31 chaperone activity

“…In agreement with crystallographic and biochemical data showing that Hsp31 relies on structural rearrangements as part of its mechanism of action at high temperatures, 18,19 we found that, although native Hsp31 was fairly resistant to degradation by PK at 37 C, its susceptibility to proteolysis was greatly increased at 45 C [ Fig. 6(A)].…”

“…were diluted into refolding buffer containing no additive or supplemented with a sixfold molar excess of clHsp31, His 6 -Hsp31, or Hsp31-His 6 at 23 C. Like native Hsp31, 13,19 cl-Hsp31 improved the recovery of active MDH by twofold and that of CS by sevenfold [ Fig. 2(A)].…”

“…Cleavage of the tag yields an Hsp31 derivative (cl-Hsp31) with 3-amino acid-long Nterminal scars (GSH) that behaves indistinguishably from authentic Hsp31 in structural and functional assays. 19 The C-terminally His-tagged variant (Hsp31-His 6 ) contains seven extra amino acids (EHHHHHH) per monomer that add %9 kDa to the molecular mass of native Hsp31. Consistent with the fact that about 1900 Å 2 of surface area are buried upon dimer formation, 14 all Hsp31 variants assembled as homodimers as judged by size exclusion chromatography and native polyacrylamide gel electrophoresis (Fig.…”

“…In light of preliminary experiments indicating that both N-and C-terminal tags influenced Hsp31 chaperone activity, we first examined the functional consequences of His-tagging on the low-temperature, housekeeping chaperone activity of Hsp31, which has been proposed to involve the binding of partially folded substrates to the Hsp31 hydrophobic bowl on the basis of mutagenesis experiments. 19 For these experiments, guanidine hydrochloride-unfolded malate dehydrogenase (MDH) or urea-unfolded citrate synthase (CS)…”

“…18,19 These amino acids form continuous hydrophobic tracts on either side of the bowl that are thought to allow high-affinity substrate binding under heat-shock conditions. Substrate ejection is believed to be driven by the return of the linker-loop ''gate'' to its original position as thermal stress subsides.…”