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Structure determination of high-energy states in a dynamic protein ensemble
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 4642617
Author(s) Stiller, John B.; Otten, Renee; Häussinger, Daniel; Rieder, Pascal S.; Theobald, Douglas L.; Kern, Dorothee
Author(s) at UniBasel Häussinger, Daniel
Year 2022
Title Structure determination of high-energy states in a dynamic protein ensemble
Journal Nature
Volume 603
Number 7901
Pages / Article-Number 528-535
Mesh terms Adenylate Kinase, metabolism; Cryoelectron Microscopy; Crystallography, X-Ray; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Thermodynamics
Abstract Macromolecular function frequently requires that proteins change conformation into high-energy states; 1-4; . 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 shifts; 5; (PCSs) with Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion; 6; (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.
Publisher Nature Publishing Group
ISSN/ISBN 0028-0836 ; 1476-4687
edoc-URL https://edoc.unibas.ch/88102/
Full Text on edoc No
Digital Object Identifier DOI 10.1038/s41586-022-04468-9
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/35236984
ISI-Number 000763347000007
Document type (ISI) Journal Article
 
   

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29/03/2024