Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90α Function in Yeast

Abstract

Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). “Closing” of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90α and hHsp90β. Although 85% identical, hHsp90β supports Hsp90 function in yeast much better than hHsp90α. Determining the basis of this difference would provide important insight into functional specificity of seemingly redundant Hsp90s, and the evolution of eukaryotic Hsp90 systems and clientele. Here, we found host co-chaperones Sba1, Cpr6 and Cpr7 inhibited hHsp90α function in yeast, and we identified mutations clustering in the N domain that considerably improved hHsp90α function in yeast. The strongest of these rescuer mutations accelerated nucleotide-dependent lid closing, N–M domain docking, and ATPase. It also disrupted binding to Sba1, which prolongs the closed state, and promoted N–M undocking and lid opening. Our data suggest the rescuer mutations improve function of hHsp90α in yeast by accelerating return to the open state. Our findings imply hHsp90α occupies the closed state too long to function effectively in yeast, and define an evolutionarily conserved region of the N domain involved in resetting the Hsp90 reaction cycle.

Nucleotide exchange is sufficient for Hsp90 functions in vivo

“…1A ). As previously observed, hHsp90α did not complement as well as hHsp90β, as seen by much weaker growth on FOA 33 – 35 . While CnHsp90 and EhHsp90 complemented S. cerevisiae Hsp90 function well, PfHsp90 complemented less well, similarly to hHsp90α.…”

“…To understand how Hsp90-EA supported growth, we used fluorescence-based techniques to investigate whether EA altered nucleotide-mediated conformational rearrangements in Hsp82, which are essential to its function in vivo. Rearrangements associated with formation of the closed-clamp such as N-M domain docking, lid repositioning and β-strand swapping are observed via photoinduced electron transfer (PET) 7 , 28 , 33 . A cysteine and a tryptophan residue are introduced at defined positions depending on which movement is to be measured.…”

“…5 ), suggesting that ATP to ADP exchange can occur in the closed-clamp conformation. This mechanism might underlie the observation that addition of ADP can quickly re-open AMP-PNP-treated Hsp90 33 . We speculate that due to Hsp90’s 2.5-fold higher affinity for ADP 53 , the cytosolic nucleotide concentration and the ATP:ADP ratio of 4:1 54 , conditions might exist on a knife’s edge where exchange in either direction can happen often enough to allow Hsp90 to function.…”

“…Recently we reported on forward mutations in human Hsp90α that improved its ability to support viability of S. cerevisiae 33 . We subsequently tested whether the forward mutations bypassed hHsp90α’s requirement for ATP hydrolysis by combining them with the hydrolysis defective mutation.…”

Visualizing the transiently populated closed-state of human HSP90 ATP binding domain

“…It was proposed that the lid acts as a nucleotide-sensitive conformational switch of the molecular chaperone activity 36 . There would be a strong correlation between the chaperone activity and the ATP vs. ADP binding 30 , 37 , 38 . In contrast, it was also reported that the transitions between the conformational states and the nucleotide binding/unbinding are mainly thermally driven, with large conformational fluctuations on timescales faster than the rate of ATP hydrolysis 39 , 40 .…”