Dataset Information

Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90? Function in Yeast.

ABSTRACT: Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). "Closing" of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90? and hHsp90?. Although 85% identical, hHsp90? supports Hsp90 function in yeast much better than hHsp90?. Determining the basis of this difference would provide important insight into functional specificity of seemingly redundant Hsp90s, and the evolution of eukaryotic Hsp90 systems and clientele. Here, we found host co-chaperones Sba1, Cpr6 and Cpr7 inhibited hHsp90? function in yeast, and we identified mutations clustering in the N domain that considerably improved hHsp90? function in yeast. The strongest of these rescuer mutations accelerated nucleotide-dependent lid closing, N-M domain docking, and ATPase. It also disrupted binding to Sba1, which prolongs the closed state, and promoted N-M undocking and lid opening. Our data suggest the rescuer mutations improve function of hHsp90? in yeast by accelerating return to the open state. Our findings imply hHsp90? occupies the closed state too long to function effectively in yeast, and define an evolutionarily conserved region of the N domain involved in resetting the Hsp90 reaction cycle.

SUBMITTER: Reidy M

PROVIDER: S-EPMC7437358 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90α Function in Yeast.

Reidy Michael M Masison Daniel C DC

Journal of molecular biology 20200619 16

Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). "Closing" of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90α and hHsp90β. Although 85% ide ...[more]

PMID: 32565117

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Project description:The SHP2 phosphatase plays a central role in a number of signaling pathways were it dephosphorylates various substrate proteins. Regulation of SHP2 activity is, in part, achieved by an intramolecular interaction between the PTP domain of the protein, which contains the catalytic site, and the N-SH2 domain leading to a "closed" protein conformation and autoinhibition. Accordingly, "opening" of the N-SH2 and PTP domains is required for the protein to become active. Binding of phosphopeptides to the N-SH2 domain is known to induce the opening event, while a number of gain-of-function (GOF) mutants, implicated in Noonan's Syndrome and childhood leukemias, are thought to facilitate opening. In the present study, a combination of computational and experimental methods are used to investigate the structural mechanism of opening of SHP2 and the impact of three GOF mutants, D61G, E76K, and N308D, on the opening mechanism. Calculated free energies of opening indicate that opening must be facilitated by effector molecules, possibly the protein substrates themselves, as the calculated free energies preclude spontaneous opening. Simulations of both wild type (WT) SHP2 and GOF mutants in the closed state indicate GOF activity to involve increased solvent exposure of selected residues, most notably Arg362, which in turn may enhance interactions of SHP2 with its substrate proteins and thereby aid opening. In addition, GOF mutations cause structural changes in the phosphopeptide-binding region of the N-SH2 domain leading to conformations that mimic the bound state. Such conformational changes are suggested to enhance binding of phosphopeptides and/or decrease interactions between the PTP and N-SH2 domains thereby facilitating opening. Experimental assays of the impact of effector molecules on SHP2 phosphatase activity against both small molecule and peptide substrates support the hypothesized mechanism of GOF mutant action. The present calculations also suggest a role for the C-SH2 domain of SHP2 in stabilizing the overall conformation of the protein in the open state, thereby aiding conformational switching between the open active and closed inactive states.

Dataset Information

Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90? Function in Yeast.

Publications

Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90α Function in Yeast.

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