Crowding Activates Heat Shock Protein 90

Abstract

Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulumspecific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro.Molecular chaperones play a central role in maintaining folded and active proteins in the cell. Hsp70 class chaperones inhibit misfolding by binding and releasing short hydrophobic segments of unstructured polypeptides with cycles of ATP hydrolysis. Hsp60 class chaperonins promote folding by isolating single protein chains within an isolated cavity. Similar to Hsp70 and Hsp60, Hsp90 has an essential ATPase activity, but the underlying functional mechanism appears to be different. Hsp90 plays important regulatory roles under non-stress conditions by its interactions with specific classes of substrates ("clients") such as kinases and nuclear receptors (1). Hsp90 has a dimeric structure that can undergo dramatic rearrangements upon ATP binding and hydrolysis. Despite significant progress in characterizing Hsp90's ATP-dependent conformational cycle in vitro, it is still unclear how Hsp90 performs its many critical cellular functions.Hsp90 conformational heterogeneity results from rigid-body rearrangements of the three domains within the monomer. The N-terminal domain (site of nucleotide binding) can rotate relative to the middle domain. The middle domain can be positioned against the C-terminal domain (site of dimerization) in multiple orientations, resulting in a flexible and structurally heterogeneous open conformation. Indeed, all full-length Hsp90 str...

Same Equilibrium. Different Kinetics. Protein Functional Consequences

“…The latter increase the affinity for N-N binding, and thus cause Hsp90 to shift from 29% closed to 46% closed population, which is in line with previous qualitative reports 41,42 . Lastly, macro-molecular crowding increased the closed population from 14% to 52%, in agreement with previous ensemble findings 43 . In contrast, to the A577I mutant, crowding slowed down fast fluctuation at the resolution limit, which creates well-separated populations in Fig.…”

In Vivo Conformational Dynamics of Hsp90 and Its Interactors

“…Taken together, these results highlight the importance of the cellular environment, where molecular crowding and protein-protein interactions play a critical role on the conformation and interactions of Hsp90 that is absent with purified Hsp90. (Ellis, 2001; Halpin et al, 2016; Kamal et al, 2003). …”

“…Furthermore, most biophysical studies on Hsp90 to date have been carried out in vitro where conditions used may perturb the natural equilibrium of populated conformers. For Hsp90, the conformation, activity and affinity for NTD inhibitors is dependent on the presence of multiple interaction partners and a crowded molecular environment (Halpin et al, 2016). In fact, Hsp90 interactions within cells are cell type-dependent (Kamal et al, 2003).…”

Hsp90 Sensitivity to ADP Reveals Hidden Regulation Mechanisms

“…Hsp90 arm closure rates measured via FRET with non-hydrolyzable ATP analogs (AMPPNP or ATP-γ-S) are often correlated with ATPase activity [18, 33, 34]. However, the rate of closure is found to be an order of magnitude slower than ATPase [17, 18].…”