Abstract
Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.
Highlights
Hsp100 chaperones cooperate with Hsp70 chaperones to disaggregate and reactivate heat-denatured proteins
Analysis of the homology model of the S. cerevisiae Hsp104 monomer indicates that the Asp-184 residue, known to be critical for prion propagation and thermotolerance, may constitute a part of an ionic interaction network
(i) Asp-184 is conserved in 100% of the analyzed sequences, (ii) basic amino acid residues are found in all sequences in the position corresponding to Lys-358, and (iii) the position corresponding to Asp-484, despite the very low overall conservation of the M domain, is occupied by an acidic amino acid in 99% of the sequences analyzed, indicating high conservation of charges at these loci (Fig. 1C)
Summary
Hsp100 chaperones cooperate with Hsp chaperones to disaggregate and reactivate heat-denatured proteins. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp chaperone in disaggregation. The importance of Hsp104 in thermotolerance is due to its ability to solubilize and refold, in cooperation with the Hsp chaperone system, proteins trapped in aggregates formed during heat stress [3] Both the disaggregation and prion propagation processes are dependent on the common mechanism of substrate threading through the central channel of Hsp104 [4, 5]. Based on the results of our previous in silico structural studies [21] and the data presented here, we put forward the hypothesis that this residue is part of a novel regulatory element of Hsp104 that consists of ionic interactions between the face-to-face surfaces of the M and NBD1 domains
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