Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

Abstract

The Hsp90 chaperone is a central node of protein homeostasis activating a large number of diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies define distinct conformational states of the mechanistic core implying structural changes that have not yet been observed in solution. Here, we engineered one-nanometer fluorescence probes based on photo-induced electron transfer into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement are mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilizes the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

Modulation of Human Hsp90α Conformational Dynamics by Allosteric Ligand Interaction at the C-Terminal Domain

“…ATP binding triggers structural rearrangements in the NTD that promote dimerization at the N-terminal, stabilizing a closed catalytically active conformation 10,19 . Transition to the closed ATPase active state is an inherently slow process recording time constants in the order of minutes 8,20,21 , possibly due to energetic barriers presented by structural intermediates that may be overcome through cofactor mediation [22][23][24][25] . ATP hydrolysis and the subsequent release of ADP from the NTD initiate a conformational return to the native apo open state and client release.…”

Hierarchical coupling between ATP hydrolysis and Hsp90’s client binding site

“…Complementary, we perform unbiased and biased all-atom MD simulations of the full dimer in a physiological NaCl solution and loadings of different nucleotides (total simulation length of 25 µs) to obtain insights into molecular mechanisms which are not directly accessible with smFRET. [36][37][38][39] We find that Arg380, which has been assumed to be involved in the functional cycle before, [40][41][42] is a prominent residue for the transfer of allosteric information from the nucleotide binding site to the full protein. Finally, we experimentally investigate both the time-and length scales of allosteric communication identified from MD simulations.…”

“…40 Furthermore, it was shown that a R380A mutant completely suppresses the formation of the closed state. 41,42 Finally, in the apo form, the binding site is at least transiently in a collapsed state, moving all mentioned protein residues closer together. Glu33 and Arg380 again are found in a salt bridge, but the position of Arg380 is significantly different from the one in nucleotide-bound state.…”

NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine

“…Recent cryo-EM structures that include co-chaperones directly highlight specific aspects of the Hsp90 conformational ensemble (Southworth and Agard 2011; Verba et al 2016). While SAXS, cryo-EM, EPR and even fluorescent energy transfer studies (Schulze et al 2016) provide valuable data on global structural forms, these are highly dynamic structures, and thus the landscape is likely further expanded through complex equilibria or multiple conformations in particular contexts. Moreover, many of the Hsp90 protein clients are themselves dynamic and/or partially folded abrogating study by crystallography and making even cryoEM challenging.…”