An interdomain sector mediating allostery in Hsp70 molecular chaperones

Smock, Robert G.; Rivoire, Olivier; Russ, William P.; Swain, Joanna F.; Leibler, Stanislas; Ranganathan, Rama; Gierasch, Lila M.

doi:10.1038/msb.2010.65

Cited by 123 publications

(203 citation statements)

References 48 publications

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“…We show that Npa3 can adopt an open state with a hydrophobic pocket, that it can bind peptides derived from Pol II subunit interfaces, that it has general chaperone activity, and that a chaperone substrate protein can stimulate its GTPase activity. The latter observation is reminiscent of the previously reported stimulation of the ATPase activity of the chaperones Hsp70 (43)(44)(45) and Hsp90 (46) by substrate binding. GTPases were also shown previously to play a key role during ribosome assembly in bacteria (47) and in eukaryotes (48).…”

Section: Discussionsupporting

confidence: 74%

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Niesser

Wagner

Kostrewa

et al. 2016

Molecular and Cellular Biology

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bBiogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

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Section: Discussionsupporting

confidence: 74%

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Niesser

Wagner

Kostrewa

et al. 2016

Molecular and Cellular Biology

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“…The fact that the hydrophobic cleft is the binding site for the Hsp70 cochaperone DnaJ, which regulates the rate of ATP hydrolysis (2,34,35), indicates that cochaperone regulation of ATPase activity most likely occurs through the same allosteric networks (i.e., via the linker and the hydrophobic cleft). Intriguingly, the hydrophobic cleft comprises a sector of coevolved residues in the NBD, which is important for stabilizing the interdomain interfaces and mediating allosteric communication between the NBD and SBD (36).…”

Section: Discussionmentioning

confidence: 99%

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

Zhuravleva

Gierasch

2011

Proc. Natl. Acad. Sci. U.S.A.

134

198

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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...

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“…The two proteins can thus effectively sequester ligands from the intracellular milieu, suggesting that they play a role in offering cytoprotection against high concentrations of bile acids. Cooperativity often arises from allosteric communication, a phenomenon that frequently escapes detection by common biochemical approaches (16). NMR and calorimetric studies performed on hI-BABP and on a series of mutants have attempted to identify the communication pathways between the two binding sites (9,10).…”

Section: Intracellular Bile Acid-binding Proteins (Babp)mentioning

confidence: 99%