Allosteric Regulation of Hsp70 Chaperones Involves a Conserved Interdomain Linker

Abstract

The 70-kDa heat shock proteins (Hsp70) are essential members of the cellular chaperone machinery that assists proteinfolding processes. To perform their functions Hsp70 chaperones toggle between two nucleotide-controlled conformational states. ATP binding to the ATPase domain triggers the transition to the low affinity state of the substrate-binding domain, while substrate binding to the substrate-binding domain in synergism with the action of a J-domain-containing cochaperone stimulates ATP hydrolysis and thereby transition to the high affinity state. Thus, ATPase and substrate-binding domains mutually affect each other through an allosteric control mechanism, the basis of which is largely unknown. In this study we identified two positively charged, surface-exposed residues in the ATPase domain and a negatively charged residue in the linker connecting both domains that are important for interdomain communication. Furthermore, we demonstrate that the linker alone is sufficient to stimulate the ATPase activity, an ability that is lost upon amino acid replacement. The linker therefore is most likely the lever that is wielded by the substrate-binding domain and the cochaperone onto the ATPase domain to induce a conformation favorable for ATP hydrolysis. Based on our results we propose a mechanism of interdomain communication.The 70-kDa heat shock protein (Hsp70) 3 chaperones assist de novo folding of newly synthesized polypeptides and disaggregation and refolding of stress-denatured proteins (1, 2). They are involved in the sorting of proteins to different cellular compartments by keeping proteins in a soluble state and assisting translocation across membranes (3-5). Hsp70s also take part in regulation of stability and activity of signal transduction proteins and assembly and disassembly of oligomeric protein structures (6). All of these functions rely on the transient interaction of the C-terminal substrate-binding domain of Hsp70 with short hydrophobic peptide stretches within the substrate polypeptides. This interaction is regulated by the nucleotide status of the ATPase domain such that the affinity of Hsp70 proteins for substrates is low when ATP is bound to their ATPase domain while the affinity is high when no nucleotide or ADP occupies the nucleotide binding pocket. In other words, ATP binding to the ATPase domain controls the conformation of the substrate-binding domain leading to prevalence for the open state of the substrate binding pocket. Vice versa, substrate binding to the substrate-binding domain in synergism with a J-domain protein induces a conformational change in the ATPase domain, thereby stimulating ATP hydrolysis. Substrate-stimulated ATP hydrolysis in return causes a conformational change in the substrate-binding domain, thereby effecting the transition to the high affinity state and the trapping of the substrate.A number of mutations in both domains of Hsp70 proteins have been isolated that interfere with this interdomain communication mechanism without providing a possible mechanism for th...

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

“…As noted above, previous biochemical studies revealed the importance of the interdomain linker for NBD conformational changes (2)(3)(4). In addition to these data, we found that the presence of the 389 VLLL 392 linker motif changes the energetics of nucleotide binding to the NBD.…”

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

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A Functional DnaK Dimer Is Essential for the Efficient Interaction with Hsp40 Heat Shock Protein

“…Previous biochemical and structural studies demonstrated that Hsp70s prefer stretches of hydrophobic segments, which normally fold inside native proteins (18 -21). A short and highly conserved linker segment, the inter-domain linker, connects NBD and SBD (22,23). Although the binding functions of NBD and SBD are independent, Hsp70 chaperone activity is strictly depen-dent on the tight coupling of these two domains upon ATP binding, which leads to modulation of the two intrinsic activities (1, 5, 24 -26).…”