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

Abstract

The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues 389 VLLL 392 ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA β-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker. T he 70-kDa heat shock proteins (Hsp70s) compose one of the most well studied and ubiquitously distributed families of allosteric proteins (1). Hsp70s assist in an extraordinarily broad spectrum of cellular processes, including protein folding, disaggregation, and translocation. All chaperone activities of Hsp70s are based on their ability to interact with short hydrophobic peptide segments of protein substrate in an ATP-dependent fashion. Hsp70s contain two domains-a 44-kDa N-terminal nucleotidebinding domain (NBD) and a 15-kDa C-terminal substrate-binding domain (SBD)-connected by a highly conserved hydrophobic linker. The allosteric cycle of Hsp70s involves an alternation between the ATP-bound state with low affinity and fast exchange rates for substrates, and the ADP-bound state with high affinity and low exchange rates for substrates. In turn, substrate binding to the SBD results in about 10-fold stimulation of ATPase activity of the NBD. However, the same ATPase stimulation can be achieved for the isolated NBD in the presence of the conserved interdomain linker sequence motif ( 389 VLLL 392 ) (2-4), indicating that the linker plays a key role in NBD function and allostery.The Hsp70 NBD belongs to the Actin/Hexokinase/Hsp70 superfamily, members of which share a number of common features (5, 6). The NBD is composed of two lobes, I and II; each lobe consists, in turn, of two subdomains: IA and IB for lobe I, and IIA and IIB for lobe II. Nucleotide binds at the bottom of the deep central cleft at the interface between subdomains IB and IIB, and all four subdomains are involved in nucleotide coordination...

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

“…Our dose responses further showed that, as suggested from the crystal structure of yeast Hsp110 with bovine Hcp70 NBD, equimolar Hsp70 and Hsp110 can optimally refold stable aggregated luciferase, likely by binding to orthologous topological sites in their respective NBDs and by effective reciprocal allosteric signals of similar quality and intensities between the two orthologous SBDs (42). The presence of such symmetrical allosteric signals was confirmed by the reciprocal release of the unfolded luciferase that we observed in the presence of apyrase, upon addition of Hsp110 to stable preformed ADP-Hsp70-Luc complexes or symmetrically of Hsp70 to stable preformed ADP-Hsp110-Luc complexes.…”

“…The chaperone mechanism of Hsp70 has been extensively studied (42). Bacterial DnaK was shown to use ATP to apply an unfolding force on stable misfolded polypeptides and small aggregates by two complementary mechanisms likely involving both direct "clamping" by individual DnaK molecules that may unfold non-native ␤-structures in stable misfolded polypeptides (12,43) and indirect "entropic pulling" by several concomitantly clamped DnaK molecules on the same misfolded polypeptide, cooperating to unfold by thermal movements local misfolded regions in between chaperone-bound segments (44).…”

Biochemical characterization of the interaction between HspA1A and phospholipids

“…Moreover, two independent reports have showed that the binding of Hsp70s to BMP and SGC Mamelak and Lingwood 2001) is mediated by amino acids different than the ones known to bind to ATP or ADP. These two arguments favor the second explanation suggesting that changes of the conformation and oligomerization protein states caused by nucleotide-binding (Young 2010;Zhuravleva and Gierasch 2011) are responsible for the observed reduction in lipid binding. The second explanation is also favored by the fact that HspA1A embeds in PS liposomes via its SBD region (Armijo et al 2014), and the finding that ATP and ADP strongly inhibit sulfatide-induced formation of the HspA1A oligomers only in the presence of KCl (Harada et al 2015).…”

Signaling through dynamic linkers as revealed by PKA

“…However, the current understanding of the allosteric role of partially unstructured linkers is at best scant. Although the role of covalent linkage in colocalization of protein domains is well known, it has recently been hypothesized that linkers, although generally quite flexible, have evolved to serve not simply as passive covalent threads connecting one domain to the next (i.e., "beadson-a-string" model), but also as active components of functionally relevant allosteric networks (22)(23)(24)(25). To test this hypothesis, here we have investigated the allosteric role of a critical linker in the regulatory subunit (R) of protein kinase A (PKA).…”