Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

Abstract

The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.

Functional characterization of natural variants found on the major stress inducible 70-kDa heat shock gene, HSPA1A, in humans

“…Based on these observations, we suggest that they might affect ATP hydrolysis by indirectly altering the bonds formed and the secondary movements of the molecule. This prediction is indirectly supported by the finding that C17, which does not bind directly to ATP, alters the ATPase activity of Hsp70 by disrupting the hydrogen-bond network within the active site [15]. Differently, the F592S mutation, which is located at the lid subdomain of the SBD and showed an increase in ADP-binding entropy [3], may alter the conformation or movement of the lid subdomain and thus indirectly affect ATP hydrolysis.…”

Integrated Dissection of Cysteine Oxidative Post-translational Modification Proteome During Cardiac Hypertrophy

“…In addition, the conserved Cys17 in the ATPase domain of the cytosolic chaperone protein, Hspa8, was continually oxidized (Cluster 7). As the perpetual oxidation of the conserved cysteine in the ER chaperone, Hspa5, has been shown to enhance chaperone function and ER homeostasis, the perpetual oxidation of Hspa8 indicates a potential function in cytosolic protein homeostasis during ISO-induced cardiac hypertrophy. ,, …”

“…As the perpetual oxidation of the conserved cysteine in the ER chaperone, Hspa5, has been shown to enhance chaperone function and ER homeostasis, the perpetual oxidation of Hspa8 indicates a potential function in cytosolic protein homeostasis during ISOinduced cardiac hypertrophy. 21,22,38 The stoichiometry is an important feature of PTM and can be calculated by comparing the abundance of a modified form with all forms of a site of interest. Integrating the temporal patterns of total cysteine abundance and reversible cysteine O-PTM further reveals the comprehensive regulation of cysteine O-PTM stoichiometry.…”

Redox regulation of yeast Hsp70 modulates protein quality control while directly triggering an Hsf1-dependent cytoprotective response

“…NEM-treated Ssa1 displayed altered trypsinization profiles and intrinsic tryptophan fluorescence indicative of structural deformation within the NBD 34 . In human Hsc70, it was found that modification of nucleotide pocket-facing C17 displaced a catalytic magnesium ion that is crucial for nucleotide hydrolysis by increasing the distance of that magnesium from several interacting residues 52 . These results are also consistent with previous data that methylene blue oxidizes cysteines 267 and 306 in the stress-inducible human Hsp70, resulting in reduced ATP binding and hydrolysis 30 .…”