Mapping the Role of Active Site Residues for Transducing an ATP-Induced Conformational Change in the Bovine 70-kDa Heat Shock Cognate Protein<sup>,</sup>

Johnson, Eric R.; McKay, David

doi:10.1021/bi990816g

Cited by 48 publications

(48 citation statements)

References 28 publications

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“…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).…”

Section: Resultssupporting

confidence: 89%

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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Section: Resultssupporting

confidence: 89%

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 proteins were heated up at a rate of 1 degree/min and the circular dichroism measured at 222 nm. At 10,20,30,37,42,50,60,70, and 85°C, circular dichroism spectra were recorded from 250 to 190 nm. The melting temperatures T m for the two temperature transition points were determined by fitting Equation 1…”

Section: Methodsmentioning

confidence: 99%

“…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 the mutual allosteric regulation of the two domains (7)(8)(9)(10)(11)(12). The recent structure of a two-domain construct of bovine Hsc70 in the nucleotide-free state shows the contact sites between the ATPase-and substrate-binding domains (13).…”

mentioning

confidence: 99%

Allosteric Regulation of Hsp70 Chaperones Involves a Conserved Interdomain Linker

Vogel

Mayer

Bukau

2006

Journal of Biological Chemistry

197

267

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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...

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“…Activities-We mutated specific residues in the NBD domain of human Hsc70 that we supposed would alter the ATPase activity of Hsc70 based on literature and structure (43)(44)(45) (Fig. 1A).…”

Section: A Human Hsc70 Variant Lacks Atpase and Refoldingmentioning

confidence: 99%

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

Fontaine

Rauch

Nordhues

et al. 2015

Journal of Biological Chemistry

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Mapping the Role of Active Site Residues for Transducing an ATP-Induced Conformational Change in the Bovine 70-kDa Heat Shock Cognate Protein^,

Cited by 48 publications

References 28 publications

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

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

Allosteric Regulation of Hsp70 Chaperones Involves a Conserved Interdomain Linker

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

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Mapping the Role of Active Site Residues for Transducing an ATP-Induced Conformational Change in the Bovine 70-kDa Heat Shock Cognate Protein,

Cited by 48 publications

References 28 publications

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

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

Allosteric Regulation of Hsp70 Chaperones Involves a Conserved Interdomain Linker

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

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Mapping the Role of Active Site Residues for Transducing an ATP-Induced Conformational Change in the Bovine 70-kDa Heat Shock Cognate Protein^,