The ATPase Cycle of the Endoplasmic Chaperone Grp94

Frey, Stephan; Leskovar, Adriane; Reinstein, Jochen; Büchner, Johannes

doi:10.1074/jbc.m704647200

Cited by 97 publications

(118 citation statements)

References 55 publications

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“…For Grp94, ATP binding proceeds via a one-step mechanism unlike other homologs, and NTD dimerization has not been directly measured. Like other homologs, the rate-limiting step is either hydrolysis or a conformational change prior to hydrolysis, and ATP is not trapped by Grp94 during hydrolysis (Frey et al, 2007). Like hHsp90 this may be due to a transient closed state.…”

Section: The Atpase Cycles Of Htpg Grp94 and Trap1mentioning

confidence: 99%

“…The same studies demonstrate that AMPPNP-bound hHsp90 remains largely in the open state and the closed state can only be observed in the presence of low levels of cross-linker. Kinetic studies suggest that the mitochondrial Trap1 is also predominantly (70%) in a closed state in the presence of ATP , whereas Grp94 remains largely in an open state (97%) in the presence of nucleotide (Frey et al, 2007). SAXS data for Grp94 confirms that it is mostly in an open, extended state in the presence of nucleotide (Krukenberg et al, in press)‥ Interestingly, bacterial HtpG has tuned the nucleotide response so that maximal levels of both the open and closed conformations are present even with saturating amounts of nucleotide Southworth & Agard, 2008).…”

Section: Hsp90 Exists In a Dynamic Equilibrium Between Conformationalmentioning

confidence: 99%

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Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

279

262

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The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1-2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organismdependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90's conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90's conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.

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Section: The Atpase Cycles Of Htpg Grp94 and Trap1mentioning

confidence: 99%

Section: Hsp90 Exists In a Dynamic Equilibrium Between Conformationalmentioning

confidence: 99%

Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

279

262

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“…GRP94 clearly binds adenosine nucleotides in vitro (22), but the low ATPase activity reported by Li and Srivastava (23) has been controversial (6,15,24). Only recently has the ATPase activity of purified GRP94 been reliably measured (25,26). These studies demonstrate that the conservation of amino acids involved in ATP binding and hydrolysis between HSP90 and GRP94 is the result of a similar, but not identical mechanism of nucleotide utilization in vitro.…”

mentioning

confidence: 93%

“…These in vivo results are best understood in light of the structural data (25) and enzymatic analysis of GRP94 (26). Both studies demonstrated that recombinant GRP94 does, in fact, possess ATPase activity, no slower than that of human HSP90␤, and that this activity is inhibited by radicicol.…”

mentioning

confidence: 98%

An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

Ostrovsky

Makarewich

Snapp

et al. 2009

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

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Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone for which only few client proteins and no cofactors are known and whose mode of action is unclear. To decipher the mode of GRP94 action in vivo, we exploited our finding that GRP94 is necessary for the production of insulin-like growth factor (IGF)-II and developed a cell-based functional assay. Grp94 ؊/؊ cells are hypersensitive to serum withdrawal and die. This phenotype can be complemented either with exogenous IGF-II or by expression of functional GRP94. Fusion proteins of GRP94 with monomeric GFP (mGFP) or mCherry also rescue the viability of transiently transfected, GRP94-deficient cells, demonstrating that the fusion proteins are functional. Because these constructs enable direct visualization of chaperone-expressing cells, we used this survival assay to assess the activities of GRP94 mutants that are defective in specific biochemical functions in vitro. Mutations that abolish binding of adenosine nucleotides cannot support growth in serumfree medium. Similarly, mutations of residues needed for ATP hydrolysis also render GRP94 partially or completely nonfunctional. In contrast, an N-terminal domain mutant that cannot bind peptides still supports cell survival. Thus the peptide binding activity in vitro can be uncoupled from the chaperone activity toward IGF in vivo. This mutational analysis suggests that the ATPase activity of GRP94 is essential for chaperone activity in vivo and that the essential protein-binding domain of GRP94 is distinct from the N-terminal domain.endoplasmic reticulum ͉ peptide hormones ͉ protein folding

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“…For example, Hsp90 inhibitors have been shown to reverse or delay the onset of tumour resistance to tyrosine kinase inhibitors, and resistance to antifungal drugs has been reversed in pre-clinical models of fungal infections (Neckers and Workman, 2012;Cowen, 2013;Miyajima et al 2013). Preliminary data suggest that chaperone-directed therapy may similarly reduce viral fitness and counteract resistance to existing antiviral therapy (Geller et al 2007). Moving forward, this represents one of the most promising clinical applications of chaperone therapeutics.…”

mentioning

confidence: 99%