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    Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry

    Year: 2021

    Journal: Biochim. Biophys. Acta-Biomembr., Volume 1863, SEP 1

    Authors: Chen, Xiaoyu; Ding, Yue; Bamert, Rebecca S.; Le Brun, Anton P.; Duff, Anthony P.; Wu, Chun-Ming; Hsu, Hsien-Yi; Shiota, Takuya; Lithgow, Trevor; Shen, Hsin-Hui

    Organizations: NHMRC [GNT1106798]; Japan Society for the Promotion of Science (JSPS) [19K16077, 18KK0197]; Institute for Fermentation Osaka; Shinnihon Foundation of Advanced Medical Treatment Research; Sasakawa Scientific Research Grant, Japan

    Keywords: Neutron reflectometry; Outer membrane proteins; Membrane biogenesis; Beta-barrel proteins

    In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex catalyses the assembly of beta-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - beta-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential beta-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Micmbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.
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