The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

Abstract

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Metadata

Item Type:	Article
Authors/Creators:	White, P https://orcid.org/0000-0001-7536-4552 Haysom, SF https://orcid.org/0000-0002-8769-090X Iadanza, MG https://orcid.org/0000-0001-6152-1844 Higgins, AJ https://orcid.org/0000-0001-8407-950X Machin, JM https://orcid.org/0000-0002-1920-149X Whitehouse, JM https://orcid.org/0000-0003-0088-4312 Horne, JE https://orcid.org/0000-0001-5260-2634 Schiffrin, B Carpenter-Platt, C Calabrese, AN https://orcid.org/0000-0003-2437-7761 Storek, KM Rutherford, ST Brockwell, DJ https://orcid.org/0000-0002-0802-5937 Ranson, NA https://orcid.org/0000-0002-3640-5275 Radford, SE https://orcid.org/0000-0002-3079-8039
Copyright, Publisher and Additional Information:	© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Dates:	Published: 7 July 2021 Published (online): 7 July 2021 Accepted: 8 June 2021
Institution:	The University of Leeds
Academic Units:	The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds)
Funding Information:	Funder Grant number Wellcome Trust 108466/Z/15/Z BBSRC (Biotechnology & Biological Sciences Research Council) BB/M012573/1 MRC (Medical Research Council) MR/P018491/1 BBSRC (Biotechnology & Biological Sciences Research Council) BB/N007603/1 BBSRC (Biotechnology & Biological Sciences Research Council) BB/T000635/1
Depositing User:	Symplectic Publications
Date Deposited:	10 Jun 2021 08:46
Last Modified:	08 Jan 2025 15:07
Status:	Published
Publisher:	Nature Research
Identification Number:	10.1038/s41467-021-24432-x
Related URLs:	Dataset
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:175072