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Rydzek, S.* ; Shein, M.* ; Bielytskyi, P.* ; Schütz, A.K.

Observation of a transient reaction intermediate illuminates the mechanochemical cycle of the AAA-ATPase p97.

J. Am. Chem. Soc. 142, 14472-14480 (2020)
Postprint DOI
Open Access Green
The human ATPase p97, also known as valosin containing protein or Cdc48, is a highly abundant AAA+ engine that fuels diverse energy-consuming processes in the human cell. p97 represents a potential target for cancer therapy and its malfunction causes a degenerative disease. Here, we monitor the enzymatic activity of p97 in real time via an NMR-based approach that allows us to follow the steps that couple ATP turnover to mechanical work. Our data identify a transient reaction intermediate, the elusive ADP.P-i nucleotide state, which has been postulated for many ATPases but has so far escaped direct detection. In p97, this species is crucial for the regulation of adenosine triphosphate turnover in the first nucleotide-binding domain. We further demonstrate how the enzymatic cycle is detuned by disease-associated gain-of-function mutations. The high-resolution insight obtained into conformational transitions in both protein and nucleotide bridges the gap between static enzyme structures and the dynamics of substrate conversion. Our approach relies on the close integration of solution- and solid-state NMR methods and is generally applicable to shed light on the mechanochemical operating modes of large molecular engines.
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Publication type Article: Journal article
Document type Scientific Article
Keywords P-31 Chemical-shifts; Conformational-changes; Thermus-thermophilus; P97/vcp Atpase; Protein; Binding; Substrate; Disease; Hydrolysis; Mechanism
ISSN (print) / ISBN 0002-7863
e-ISSN 1520-5126
Quellenangaben Volume: 142, Issue: 34, Pages: 14472-14480 Article Number: , Supplement: ,
Publisher American Chemical Society (ACS)
Publishing Place 1155 16th St, Nw, Washington, Dc 20036 Usa
Reviewing status Peer reviewed
Grants Bavarian NMR Centre
CIPSM DFG cluster
Fonds der Chemischen Industrie