Hsp70 Chaperone Ligands Control Domain Association via an Allosteric Mechanism Mediated by the Interdomain Linker

Abstract

Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.

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

“…5A) shows that most resonances do not move in a simple manner in response to ligands, and thus suggests that ligands adjust the populations of more than two NBD conformations. The conformational heterogeneity for the Hsp70 NBD ensemble implied by the chemical-shift behavior is consistent with computational predictions (11) and previous experiments (3,9,10,27). These observations also account for previous results showing that changes in the nucleotide-binding site, such as single point mutations or the absence of inorganic ions, cause disruption of Hsp70 allostery without significant changes in structure by X-ray crystallography (1,20,21,26,28,29).…”

“…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.…”

“…On the contrary, when the 389 VLLL 392 motif is present on the isolated NBD, ATP binds more tightly than ADP, mimicking behavior of the full-length protein with K d values of 250 and 160 nM for ADP and ATP, respectively (23). Moreover, NMR evidence gathered using the NBD 392 construct (residues 1-392) pointed to a coupling of the effects of the linker and the nucleotide on NBD conformation (3 To explore the structural features that enable the NBD to bind and release nucleotide, as required in its allosteric cycle, we analyzed chemical-shift perturbations between the nucleotide-free and ADPbound states of the NBD; the data are presented as a function of residue position in Fig. 1A.…”

“…Plasmids encoding DnaK NBD(1-388) T199A, NBD(1-392)T199A, and NBD(1-393)T199A were prepared previously in our laboratory; they were expressed in E. coli and purified as described (3,37). 2 H-, 13 C-, and 15 N-labeled NMR samples were prepared using established protocols (38), as described previously (37) (see SI Text).…”

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

“…The bacterial chaperone DnaK has long served as a prototype for the study of Hsp70 chaperones and its investigation provided important insights into the structure and function of this large group of proteins (Swain et al 2007). Three types of chaperone activities have been described for bacterial DnaK: (1) passively preventing the aggregation of artificially unfolded proteins (holding function).…”