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Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter

The Amino Acid–Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, con...

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Autores principales: del Alamo, Diego, DeSousa, Lillian, Nair, Rahul M., Rahman, Suhaila, Meiler, Jens, Mchaourab, Hassane S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9407458/
https://www.ncbi.nlm.nih.gov/pubmed/35969794
http://dx.doi.org/10.1073/pnas.2206129119
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author del Alamo, Diego
DeSousa, Lillian
Nair, Rahul M.
Rahman, Suhaila
Meiler, Jens
Mchaourab, Hassane S.
author_facet del Alamo, Diego
DeSousa, Lillian
Nair, Rahul M.
Rahman, Suhaila
Meiler, Jens
Mchaourab, Hassane S.
author_sort del Alamo, Diego
collection PubMed
description The Amino Acid–Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold.
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spelling pubmed-94074582023-02-15 Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter del Alamo, Diego DeSousa, Lillian Nair, Rahul M. Rahman, Suhaila Meiler, Jens Mchaourab, Hassane S. Proc Natl Acad Sci U S A Biological Sciences The Amino Acid–Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold. National Academy of Sciences 2022-08-15 2022-08-23 /pmc/articles/PMC9407458/ /pubmed/35969794 http://dx.doi.org/10.1073/pnas.2206129119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
del Alamo, Diego
DeSousa, Lillian
Nair, Rahul M.
Rahman, Suhaila
Meiler, Jens
Mchaourab, Hassane S.
Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title_full Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title_fullStr Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title_full_unstemmed Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title_short Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter
title_sort integrated alphafold2 and deer investigation of the conformational dynamics of a ph-dependent apc antiporter
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9407458/
https://www.ncbi.nlm.nih.gov/pubmed/35969794
http://dx.doi.org/10.1073/pnas.2206129119
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