Linking function to global and local dynamics in an elevator-type transporter

Didar Ciftci; Chloe Martens; Vishnu G. Ghani; Scott C. Blanchard; Argyris Politis; Gerard H.M. Huysmans; Olga Boudker

doi:10.1073/pnas.2025520118

Original language	English
Article number	e2025520118
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	118
Issue number	49
DOIs	https://doi.org/10.1073/pnas.2025520118
Published	7 Dec 2021
Additional links	Link to publication in Scopus

Bibliographical note

Funding Information: We thank Roger Altman for the preparation of smFRET chambers, Daniel S. Terry for providing access to the TIRF microscopy setup for single-molecule imaging, and William Eng for assistance with protein production. Funding was provided by The National Institute of Neurological Disorders and Stroke (NINDS) Grants R37NS085318 and R01NS111767 to O.B., American Heart Association (AHA) Fellowship 19PRE34380215 to D.C., and Grant 7R01GM098859 to S.C.B. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Maire Sklodowska-Curie Grant Agreement MEMDYN 660083 (G.H.M.H.). A.P.?s work was supported by the Wellcome Trust Grant (109854/Z/15/Z) and the Leverhulme Trust Grant (RPG-2019-178). C.M. is a Research Fellow of the Fonds National de la Recherche Scientifique (FRS-FNRS, Belgium). Funding Information: Data Availability. All data needed to evaluate the conclusions in the paper are included in the article and SI Appendix. Raw data for single molecule FRET trajectories can be found in GitHub, https://github.com/BoudkerLab/PNAS_data Uptake plots of the HDX-MS data can be accessed on Figshare (DOIs: 10.6084/ m9.figshare.16825315; 10.6084/m9.figshare.16825306; 10.6084/m9.figshare. 16825300) ACKNOWLEDGMENTS. We thank Roger Altman for the preparation of smFRET chambers, Daniel S. Terry for providing access to the TIRF microscopy setup for single-molecule imaging, and William Eng for assistance with protein production. Funding was provided by The National Institute of Neurological Disorders and Stroke (NINDS) Grants R37NS085318 and R01NS111767 to O.B., American Heart Association (AHA) Fellowship 19PRE34380215 to D.C., and Grant 7R01GM098859 to S.C.B. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Maire Sklodowska-Curie Grant Agreement MEMDYN 660083 (G.H.M.H.). A.P.’s work was supported by the Wellcome Trust Grant (109854/Z/15/Z) and the Leverhulme Trust Grant (RPG-2019-178). C.M. is a Research Fellow of the Fonds National de la Recherche Scientifique (FRS-FNRS, Belgium). Publisher Copyright: © 2021 National Academy of Sciences. All rights reserved.

Abstract

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

King's College London

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