Structure of a nascent membrane protein as it folds on the BAM complex

Abstract

Mitochondria, chloroplasts, and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure 1 , 2 . β-barrels are sheets of β-strands wrapped into a cylinder with the first strand hydrogen-bonded to the last strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane 3 – 5 . One subunit of the machines is itself a β-barrel protein that plays a central role in folding other β-barrels. In Gram-negative bacteria, the β- b arrel a ssembly m achine (Bam) consists of the β-barrel protein BamA and four lipoproteins 5 – 8 . To understand how the Bam complex accelerates folding without using exogenous energy (e.g., ATP) 9 , we trapped folding intermediates on the machine. We report here the structure of the Bam complex folding BamA itself. The BamA catalyst (BamA M , for BamA machine ) forms an asymmetric hybrid β-barrel with the BamA substrate (BamA S ). The N-terminal edge of BamA M has an antiparallel hydrogen-bonded interface with the C-terminal edge of BamA S , consistent with previous crosslinking studies 10 – 12 ; the other edges of BamA M and BamA S are close to each other but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding but creates a high kinetic barrier to substrate release once folding has finished. Features at each end of the substrate overcome the barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the Bam complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

“…This structure shows a late-stage assembly intermediate and, along with the cross-linking data, appears to support model 3 (BamA-swing/elongation) or 4 (BamA lumen-catalyzed) of BAM-catalyzed OMP folding shown in Fig. 7 ( 403 ).…”

The Extracellular Loops of OmpA Control the Slow Rate ofIn VitroFolding

“…We anticipate that this is consistent with the conclusions of previous work that it may be the turns and not the loops that facilitate the zipping (Danoff and Fleming 2017). However, the lack of importance of loops to the folding rate may not be universal for all OMPs as shortening of the loops of BamA lead to systematic slowing of its folding (Tomasek, et al 2020). More work will need to be done to determine the universality of the relative importance of the loops and turns in folding.…”

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

“…Numbering of residue positions in the last β-strand (the β signal) follows a publication by Robert et al 36 In line with this, residues F802-I806 of BamA (substrate) form an anti-parallel strand with β1 BamA (of BAM complex) as revealed by the recent cryo-EM structure of a substrate trapped on the BAM complex that snapshots a later intermediate stage of β-OMP biogenesis. 43 To rule out the possibility that the impaired growth of the E coli strain JCM320 resulted from their decreased protein expression levels caused by mutation, we carried out western blotting to compare the expression levels of these mutants with that of the wild-type BamA. As show in Figures 4A and S6A (low panel), mutation itself did not cause a significant variation of the protein expression levels of BamA mutants.…”

“…36,42 Recently, a breakthrough has been made by cryo-EM structure determination of a folding intermediate trapped on the BAM complex, snapshotting an important step in β-OMP biogenesis. 43 Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex bound with different substrates. Structural analysis indicates that membrane composition surrounding the BAM complex could modulate the overall conformation of the BAM complex, further highlighting low energy barriers among different conformational states.…”