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

Abstract

HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear. Here, we identify a metastable excited state in the isolated human HSP90α ATP binding domain. We use solution NMR and mutagenesis to characterize structures of both ground and excited states. We demonstrate that in solution the HSP90α ATP binding domain transiently samples a functionally relevant ATP-lid closed state, distant by more than 30 Å from the ground state. NMR relaxation enables to derive information on the kinetics and thermodynamics of this interconversion, while molecular dynamics simulations establish that the ATP-lid in closed conformation is a metastable exited state. The precise description of the dynamics and structures sampled by human HSP90α ATP binding domain provides information for the future design of new therapeutic ligands.

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“…This suggests that residues 104–114 of the ATP-lid in apo N-HSP90α exist in different loop conformations in solution. The binding of specific ligands, different crystallization conditions, additives, pH, and other factors can alter the pre-existing conformational equilibrium. , This leads to a shift in the population distribution toward more stable conformations, thereby increasing the proportion of this conformation. In the isolated structure of N-HSP90α, Henot et al also observed a “closed” conformation of the ATP-lid, with an occupancy of 3–4% at room temperature .…”

“…The binding of specific ligands, different crystallization conditions, additives, pH, and other factors can alter the pre-existing conformational equilibrium. , This leads to a shift in the population distribution toward more stable conformations, thereby increasing the proportion of this conformation. In the isolated structure of N-HSP90α, Henot et al also observed a “closed” conformation of the ATP-lid, with an occupancy of 3–4% at room temperature . However, this helical conformation remains elusive in the absence of ligand binding, likely triggered by specific binding events.…”

“…N-HSP90α has a relatively flexible ATP-binding pocket. Henot et al clustered over 300 atomic-resolution structures of N-HSP90α and classified them into eight classes based on a threshold . In this study, we focused on significant conformational differences in the region of residues 104–114, as it profoundly influences the volume and solvent distribution of the ATP-binding pocket, impacting the binding kinetics behavior of ligands.…”

“…The ATP pocket comprises a highly flexible lid region (residues 102–137) that undergoes remarkable conformational changes during the functional cycle of the chaperone, characterized by the opening and closing of the lid. Importantly, this conformational change is not exclusively determined by client protein and nucleotide binding. , Using solution nuclear magnetic resonance spectroscopy, Henot et al identified the closed conformation of apo N-HSP90α . The residues 104–114 of the ATP-lid are highly plastic and exhibit different “loop-out” and “loop-in” conformations in the absence of a bound ligand .…”

“… 50 , 51 Using solution nuclear magnetic resonance spectroscopy, Henot et al identified the closed conformation of apo N-HSP90α. 52 The residues 104–114 of the ATP-lid are highly plastic and exhibit different “loop-out” and “loop-in” conformations in the absence of a bound ligand. 53 When a ligand is bound, new “loop-middle” and “helical” conformations are formed.…”