The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells
The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells
Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.
Galazzo, Laura
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Meier, Gianmarco
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Januliene, Dovile
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Parey, Kristian
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De Vecchis, Dario
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Striednig, Bianca
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Hilbi, Hubert
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Schäfer, Lars V.
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Kuprov, Ilya
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Moeller, Arne
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Bordignon, Enrica
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Seeger, Markus A.
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12 October 2022
Galazzo, Laura
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Meier, Gianmarco
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Januliene, Dovile
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Parey, Kristian
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De Vecchis, Dario
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Striednig, Bianca
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Hilbi, Hubert
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Schäfer, Lars V.
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Kuprov, Ilya
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Moeller, Arne
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Bordignon, Enrica
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Seeger, Markus A.
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Galazzo, Laura, Meier, Gianmarco, Januliene, Dovile, Parey, Kristian, De Vecchis, Dario, Striednig, Bianca, Hilbi, Hubert, Schäfer, Lars V., Kuprov, Ilya, Moeller, Arne, Bordignon, Enrica and Seeger, Markus A.
(2022)
The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells.
Science Advances, 8 (41).
(doi:10.1126/sciadv.abn6845).
Abstract
Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.
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sciadv.abn6845
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Accepted/In Press date: 24 August 2022
Published date: 12 October 2022
Additional Information:
We acknowledge B. Blattmann and C. Stutz-Ducommun of the Protein Crystallization Center UZH for performing the crystallization screening and the staff of the SLS beamline X06DA for the support during data collection. We thank S. Štefanić of the Nanobody Service Facility (NSF), University of Zürich, for alpaca immunization. We thank C. von Ballmoos for giving technical advice on how to measure ATP/ADP ratios. We are grateful to W. Kühlbrandt and staff for access to the cryo-EM facility at the Max Planck Institute of Biophysics.
Funding: Research in the laboratory of I.K. was supported by grants from Leverhulme Trust (RPG-2019-048) and MathWorks and used NVIDIA Tesla A100 GPUs through NVIDIA Academic Grants Programme. Research in the laboratory of A.M. was funded by DFG through CRC944. Research in the laboratory of E.B. and L.V.S. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy-EXC 2033-390677874–RESOLV and grant SCHA 1574/6-1. Research in the laboratory of M.A.S. was funded by the European Research Council (ERC) (consolidator grant no. 772190), an SNSF Professorship of the Swiss National Science Foundation (PP00P3_144823), and a project grant of the Swiss National Science Foundation (310030_188817).
Identifiers
Local EPrints ID: 476593
URI: http://eprints.soton.ac.uk/id/eprint/476593
ISSN: 2375-2548
PURE UUID: 872ef359-ad56-4bfa-8c4d-edcc6a5b104d
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Date deposited: 09 May 2023 16:57
Last modified: 17 Mar 2024 03:28
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Contributors
Author:
Laura Galazzo
Author:
Gianmarco Meier
Author:
Dovile Januliene
Author:
Kristian Parey
Author:
Dario De Vecchis
Author:
Bianca Striednig
Author:
Hubert Hilbi
Author:
Lars V. Schäfer
Author:
Arne Moeller
Author:
Enrica Bordignon
Author:
Markus A. Seeger
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