The Hsp90 Chaperone: ¹H and ¹⁹F Dynamic Nuclear Magnetic Resonance Spectroscopy Reveals a Perfect Enzyme

Abstract

Hsp90 is a crucial chaperone whose ATPase activity is fundamental for stabilizing and activating a diverse array of client proteins. Binding and hydrolysis of ATP by dimeric Hsp90 drive a conformational cycle characterized by fluctuations between a compact, N-and C-terminally dimerized catalytically competent closed state and a less compact open state that is largely C-terminally dimerized. We used 19 F and 1 H dynamic nuclear magnetic resonance (NMR) spectroscopy to study the opening and closing kinetics of Hsp90 and to determine the k cat for ATP hydrolysis. We derived a set of coupled ordinary differential equations describing the rate laws for the Hsp90 kinetic cycle and used these to analyze the NMR data. We found that the kinetics of closing and opening for the chaperone are slow and that the lower limit for k cat of ATP hydrolysis is ∼1 s −1 . Our results show that the chemical step is optimized and that Hsp90 is indeed a "perfect" enzyme.

Controlling protein function by fine-tuning conformational flexibility

“…Both global arrangements are semi-stable at room temperature. As a consequence, Hsp90 alternates constantly between open and closed conformations - even in the absence of the chemical energy source, ATP ( Mickler et al, 2009 ; Schmid et al, 2016 ; Lee et al, 2019 ). Surprisingly, the characteristic conformational changes of Hsp90 are only little affected by e.g.…”

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.…”

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels

“…This would be equally valuable in, e.g., probing of enzymatic interactions, or in any situation where characterization of intermolecular interactions might be confounded by overlap of chemical shifts between species. Solution-state 19 F NMR spectroscopy may notably be applied even in situations with relatively high molecular weight/slow tumbling (e.g., a recent comprehensive enzyme kinetics characterization of a ~90 kDa Hsp90 enabled by 19 F NMR methods [44] or demonstration of a new 19 F-13C transverse relaxation optimized spectroscopy (TROSY) NMR methodology capable of reporting on a ~180 kDa α7 single-ring of a 20S proteasome core particle [45]). This adds to the versatility and cost-effectiveness of this technique relative to, e.g., the requirement to deuterate the target protein and specifically introduce 1 H at desired probe sites.…”