Structure determination of high-energy states in a dynamic protein ensemble

John B. Stiller, Renee Otten, Daniel Häussinger, Pascal S. Rieder, Douglas L. Theobald and Dorothee Kern ()
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John B. Stiller: Brandeis University
Renee Otten: Brandeis University
Daniel Häussinger: University of Basel
Pascal S. Rieder: University of Basel
Douglas L. Theobald: Brandeis University
Dorothee Kern: Brandeis University

Nature, 2022, vol. 603, issue 7901, 528-535

Abstract: Abstract Macromolecular function frequently requires that proteins change conformation into high-energy states1–4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion6 (PCS–CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS–CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.

Date: 2022
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DOI: 10.1038/s41586-022-04468-9

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