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Structural Dynamics of the Hsp90 Chaperone from 19F NMR spectroscopy
- Author / Creator
- Rashid, Suad
Living cells have evolved a complex machinery of molecular chaperones to protect proteins from misfolding/aggregation in the protein-rich intracellular environment. Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone ubiquitously expressed in eubacteria and eukaryotes. The chaperone has been implicated in activating and stabilizing a diverse range of protein substrates (clients), including protein kinases, transcription factors, and nuclear hormone receptors. The diverse clients of Hsp90 are involved in the regulation of vital cellular processes such as cell survival and proliferation, transcriptional regulation, apoptosis, gene expression, and signal transduction. This makes Hsp90 an essential protein for maintaining cellular protein homeostasis.
Hsp90 is a homodimer with each protomer consisting of an N-terminal domain (N), a middle domain (M), and a C-terminal domain (C), forming the dimerization interface of the homodimer. The chaperone activity of Hsp90 is accompanied by a series of conformational alterations, which transforms Hsp90 from an N-terminally open conformation to a catalytically active closed conformation, bearing interprotomer N-N domain and intraprotomer N-M domain associations. Importantly, the rate of acquisition of this catalytically active closed conformation is slow, thus the rate-limiting step of the Hsp90 chaperone cycle. The overall conformational change of Hsp90 from an open to a closed conformation is driven by local transitions that are mainly localized within the N-terminal domain of Hsp90. In this work, we used a combination of 19F NMR-based techniques and molecular dynamics simulations to investigate the dynamics of local and global motions occurring within the Hsp90 chaperone.
To facilitate a quantitative understanding of exchange contributions to the 19F NMR linewidth of Hsp90, we quantified the internal motion of the sidechain of a trifluoroacetone cysteine derivative (CYF) commonly employed in 19F NMR studies as a reporter of dynamic processes occurring in proteins. Our results show that the high flexibility of the CYF sidechain contributes to the narrow 19F NMR linewidths of CYF tagged proteins and demonstrate the suitability of the CYF probe for studying dynamics occurring in large molecular weight proteins. The quantification of CYF sidechain motion allowed us to explore ATP-lid dynamics as well as the kinetics of nucleotide binding to the N-terminal domain of Hsp90. Our findings reveal priming of the ATP-lid by the -phosphate of nucleotide (ATP/AMPPNP), which is a prerequisite for full ATP-lid closure and subsequent ATP hydrolysis by Hsp90. Also, we investigated the dynamics of full-length Hsp90 in the presence of different nucleotides and the ATPase stimulating co-chaperone Aha1 using 19F NMR as a reporter of conformational transitions. We found that binding of AMPPNP to full-length Hsp90 induces equilibrium populations of open and closed conformations of Hsp90 and Aha1 accelerates the slow conformational changes that lead to the formation of the closed conformation. Taken together, the research presented here highlight the remarkable utility of 19F NMR spectroscopy for investigating local dynamics occurring within the N-terminal domain of Hsp90 and provides the basis for studying both local and global dynamics of the full-length chaperone in the presence of nucleotides, co-chaperones, and clients.
- Graduation date
- Fall 2021
- Type of Item
- Doctor of Philosophy
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