Hsp110 Is a Nucleotide-activated Exchange Factor for Hsp70

Abstract

Members of the abundant Hsp70 class of chaperones play essential roles in diverse cellular processes, including the folding and assembly of newly synthesized proteins, the transport of proteins across membranes, the refolding of misfolded proteins, and the regulation of signal transduction proteins (1). Hsp70 chaperones transiently associate with peptide segments of protein substrates, thereby affecting their folding (2). The binding and release of substrates is driven by the ATPase cycle of the Hsp70. ATP binding to the N-terminal nucleotide-binding domain (NBD) 6 induces conformational changes in the C-terminal substrate-binding domain that result in low affinity for substrates and high binding and release rates. ADP on the other hand results in higher affinity for substrates and low binding/release rates and thereby causes the trapping of substrates.The basic chaperone cycle of Hsp70 proteins is modulated by cofactors that affect either ATP hydrolysis or exchange of ADP for ATP. The hydrolysis of ATP is stimulated by J-domainharboring co-chaperones that also deliver substrates and thereby facilitate substrate binding as well as provide specificity to the Hsp70s. Nucleotide exchange factors (NEFs) accelerate nucleotide release by associating with specific conformations of the Hsp70 NBD that exhibit low affinity for ADP and ATP. Rebinding of ATP and the concomitant dissociation of the NEF complete the exchange reaction.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm

“…Human Hsp110 (HSPA4L) was produced as a 6His-SUMO-2G-HSPA4L fusion in E. coli and purified on Talon resin. The eluted protein was treated with purified ULP1-His to cleave the SUMO moiety, and the HSPA4L was recovered free of both His-SUMO and ULP1-His by passage through Talon resin (32,44).…”

Insights into the structural dynamics of the Hsp110–Hsp70 interaction reveal the mechanism for nucleotide exchange activity

“…The Hsp70 NBD has been shown to be sufficient for interaction, whereas on the Hsp110 side both the NBD and SBD could be involved (11,12). Sse1 is also activated by ATP.…”

“…Sse1 is also activated by ATP. Upon ATP binding the protein adopts a stabilized conformation that is required for association with its cognate Hsp70, Ssa1 (12). However, after the complex has formed, nucleotide can be completely removed from Sse1 without any perturbation of the integrity of the complex.…”

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

“…1B), indicating a substantial stabilization of Ssz by ATP. Under these conditions, the exchange rate corresponds to that observed for canonical Hsp70 chaperones like DnaK (15,20). The nucleotide-induced stabilization, however, vanished with longer incubation times in D 2 O, implying that the nucleotide is released within minutes.…”

“…This unusually fast exchange kinetics is indicative of a highly dynamic and loosely folded protein conformation. Because nucleotide binding affects the conformational dynamics of other Hsp70 family members (15,19,20), we added 0.6 mM ATP to the reaction. The presence of ATP resulted in strongly decreased deuteron incorporation to 45% after a 2-min HX reaction (Fig.…”