Structures of the β‐barrel assembly machine recognizing outer membrane protein substrates

Abstract

β‐barrel outer membrane proteins (β‐OMPs) play critical roles in nutrition acquisition, protein import/export, and other fundamental biological processes. The assembly of β‐OMPs in Gram‐negative bacteria is mediated by the β‐barrel assembly machinery (BAM) complex, yet its precise mechanism remains elusive. Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex with bound substrates. Structural analysis indicates that the membrane compositions surrounding the BAM complex could modulate its overall conformations, indicating low energy barriers between different conformational states and a highly dynamic nature of the BAM complex. Importantly, structures of the BAM complex with bound substrates and the related functional analysis show that the first β‐strand of the BamA β‐barrel (β1BamA) in the BAM complex is associated with the last but not the first β‐strand of a β‐OMP substrate via antiparallel β‐strand interactions. These observations are consistent with the β‐signal hypothesis during β‐OMP biogenesis, and suggest that the β1BamA strand in the BAM complex may interact with the last β‐strand of an incoming β‐OMP substrate upon their release from the chaperone‐bound state.

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

“…The difference between eL1 conformations in the two BAM-LL structures is striking, and suggests that this region must be highly malleable to allow disulfide bond formation within the BamA β-barrel. Interestingly, the 'contorted open' BAM-LL structure closely resembles a recent structure of WT BAM in saposin nanodiscs 22 in which eL1 adopts this inward conformation in the absence of disulfide tethering. In accord with this idea, eL1 can adopt a wide range of conformations in lateralopen BAM structures (Supplementary Fig.…”

“…Here, we have used structural, biochemical and kinetic refolding analyses to dissect these two roles, at least for the 8-stranded OMPs, tOmpA and OmpX. BAM is well-known to be conformationally dynamic, with cryo-EM and X-ray structures capturing the complex in lateral-open 5,6,8 and lateral-closed 6,7,21,22 conformations, and a recent cryoEM, MD and single-molecule FRET study demonstrating dynamics of the complex in nanodiscs 18 . Furthermore, recent X-ray structures have demonstrated that the C-terminal strand of the OMP substrates OmpA and OmpLA forms an antiparallel β-strand pairing with lateralclosed BamA β1, possibly capturing an early stage intermediate in OMP assembly 22 .…”

“…BAM is well-known to be conformationally dynamic, with cryo-EM and X-ray structures capturing the complex in lateral-open 5,6,8 and lateral-closed 6,7,21,22 conformations, and a recent cryoEM, MD and single-molecule FRET study demonstrating dynamics of the complex in nanodiscs 18 . Furthermore, recent X-ray structures have demonstrated that the C-terminal strand of the OMP substrates OmpA and OmpLA forms an antiparallel β-strand pairing with lateralclosed BamA β1, possibly capturing an early stage intermediate in OMP assembly 22 . A recent cryoEM structure of a BAM:tBamA complex revealed that the tBamA substrate forms a β-strand pairing with lateral-open BamA β1 of BAM, whilst making a sidechain mediated interface involving BamA β16, to form a hybrid barrel 29 that presumably mimics a late-stage assembly intermediate.…”

“…In the 'lateral-open' conformation, as captured by the cryoEM structure of the intact complex 8 and X-ray crystallography of the BamACDE sub-complex 5,6 , β1 and β16 are separated. In contrast, crystal structures of the intact BAM complex are in a 'lateral-closed' conformation both in the absence 6,7 or presence of peptide fragments of substrate 21,22 , wherein β1 and β16 are hydrogen-bonded, albeit with fewer hydrogen bonds than exist between the other strands in the barrel 1 . The POTRA domains are also dynamic, with motions of POTRA-5 also occurring alongside changes in gate conformation, with POTRA-5 plugging the entrance to the BamA β-barrel lumen in the lateral-open structures, but moving aside when the lateral gate is closed 18 .…”

“…Firstly conformational changes in BAM, and protein-protein interactions between BAM and substrate OMPs are thought to be involved in catalysing folding [25][26][27][28][29] . These models all involve a folding intermediate in which the C-terminal β-strand of the substrate is associated with BamA-β1, as supported by crosslinking 26,27 , a recent cryoEM structure of a hybrid barrel formed between BAM and tBamA (the transmembrane domain of a BamA substrate) 29 , and crystal structures of BAM covalently tethered to the Cterminal β-strands of OMP substrates OmpA and OmpLA 22 . Variations of these models include the 'barrel elongation' 25 and 'swing' 27 models which suggest that folding begins in the periplasm, and also 'budding' models 1,3,25 wherein OMPs are thought to enter the lumen of the BamA barrel and fold via sequential addition of β-hairpin units 26 .…”