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Mechanism of the Hsp90 chaperone cycle: investigation of divalent ion binding and conformational change

Patel, D.; (2012) Mechanism of the Hsp90 chaperone cycle: investigation of divalent ion binding and conformational change. Doctoral thesis , UCL (University College London). Green open access


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Hsp90 chaperones a large number of client proteins that perform diverse functions including roles in signal transduction and cell cycle regulation. Since a subset of these clients are proto-oncogenes, Hsp90 has emerged as a target for anti-cancer therapeutics. The chaperone function of Hsp90 involves an ATP-dependent cycle of conformational changes: recently, X-ray crystallography and fluorescence resonance energy transfer (FRET) have provided evidence that these changes occur primarily within the N-terminal and middle domains of the protein which are thought to form a closed conformation following ATP binding that facilitates ATP hydrolysis. The research described herein is broadly divided into two sections. Initially we investigate the thermodynamics of nucleotide binding to the isolated N-terminal domain of Hsp90 (N-Hsp90) and the role of the divalent Mg2+ ion in this interaction. This provides a rationale for how the binding of two structurally similar nucleotides, AMP-PNP (Adenosine 5′-(β,γ-imido)triphosphate tetralithium salt hydrate); an ATP analogue) and ADP that differ in structure by a single phosphate group, produce significantly different thermodynamic signatures. Isothermal titration calorimetry was used to characterize the thermodynamic profiles of nucleotide binding to N-Hsp90 and various mutants of NHsp90. The effect of incorporation of another divalent ion when compared to the native Mg2+ ion in the binding of the AMP-PNP ligand has a significant effect on the thermodynamic properties of complex formation. By substituting different cations in the solvent we were able to establish the potential importance of the hydration properties of the metal ion in the associated observed heat capacities and resulting conformational changes of the domain. Secondly, we investigated the conformational changes associated with the turnover of ATP by the full length Hsp90 protein. It is well know that the conformational changes that take place post ATP binding and hydrolysis are responsible for the activation of Hsp90 and its role in the chaperoning of the large plethora of clientele that it has been associated with. Investigating the nature of the conformational changes associated with the full length Hsp90 during its ATP cycle using double electron-electron resonance (DEER) spectroscopy revealed previously unknown dynamics of the chaperone even in the absence of AMP-PNP. Furthermore, this method also revealed additional rearrangements whereby all three domains of Hsp90 undergo opening and closing in the absence and presence of the nucleotide. Furthermore, the crystal structure of the full length Hsp90 in complex with AMP-PNP and the co-chaperone Sba1/p23 revealed a conformation where the M domains of the dimer are shown to be in close association to one another; our data suggests that this state is only achieved upon the binding of the co-chaperone Sba1/p23, and not as a result of AMP-PNP binding as has previously been suggested. The findings from the DEER data provide a potential mechanism in which Sba1/p23 ‘halts’ the ATP cycle of Hsp90 in an ATP ‘active’ state, thereby enabling Sba1/p23 to recruit and process its client proteins. The additional conformational rearrangements observed in our study could be viewed as potential targets for the development of innovative therapeutics against this highly diverse molecular chaperone.

Type: Thesis (Doctoral)
Title: Mechanism of the Hsp90 chaperone cycle: investigation of divalent ion binding and conformational change
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1348542
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