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

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 19F and 1H dynamic nuclear magnetic resonance (NMR) spectroscopy to study the opening and closing kinetics of Hsp90 and to determine the kcat 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 kcat of ATP hydrolysis is ∼1 s-1. Our results show that the chemical step is optimized and that Hsp90 is indeed a "perfect" enzyme.

Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field 19F NMR Spectroscopy.

Protein turnover in cells is regulated by the ATP dependent activity of the Hsp90 chaperone. In concert with accessory proteins, ATP hydrolysis drives the obligate Hsp90 dimer through a cycle between open and closed states that is critical for assisting the folding and stability of hundreds of proteins. Cycling is initiated by ATP binding to the ATPase domain, with the chaperone and the active site gates in the dimer in open states. The chaperone then adopts a short-lived, ATP bound closed state with a closed active site gate. The structural and dynamic changes induced in the ATPase domain and active site gate upon nucleotide binding, and their impact on dimer closing are not well understood. We site-specifically 19F-labeled the ATPase domain at the active site gate to enable benchtop and high field 19F NMR spectroscopic studies. Combined with MD simulations, this allowed accurate characterization of pico- to nanosecond time scale motions of the active site gate, as well as slower micro- to millisecond time scale processes resulting from nucleotide binding. ATP binding induces increased flexibility at one of the hinges of the active site gate, a necessary prelude to release of the second hinge and eventual gate closure in the intact chaperone.

Side-Chain Dynamics of the Trifluoroacetone Cysteine Derivative Characterized by 19F NMR Relaxation and Molecular Dynamics Simulations.

19F NMR spectroscopy is a powerful tool for the study of the structures, dynamics, and interactions of proteins bearing cysteine residues chemically modified with a trifluoroacetone group (CYF residue). 19F NMR relaxation rates for the fluoromethyl group of CYF residues are sensitive to overall rotational tumbling of proteins, fast rotation about the CF3 methyl axis, and the internal motion of the CYF side-chain. To develop a quantitative understanding of these various motional contributions, we used the model-free approach to extend expressions for 19F- T2 NMR relaxation to include side-chain motions for the CYF residue. We complemented the NMR studies with atomic views of methyl rotation and side-chain motions using molecular dynamics simulations. This combined methodology allows for quantitative separation of the contributions of fast pico- to nanosecond dynamics from micro- to millisecond exchange processes to the 19F line width and highlights the utility of the CYF residue as a sensitive reporter of side-chain environment and dynamics in proteins.