The β-barrel assembly machinery in motion

Abstract

In Gram-negative bacteria, the biogenesis of β-barrel outer membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex. During the past decade, structural and functional studies have collectively contributed to advancing our understanding of the structure and function of the BAM complex; however, the exact mechanism that is involved remains elusive. In this Progress article, we discuss recent structural studies that have revealed that the accessory proteins may regulate essential unprecedented conformational changes in the core component BamA during function. We also detail the mechanistic insights that have been gained from structural data, mutagenesis studies and molecular dynamics simulations, and explore two emerging models for the BAM-mediated biogenesis of OMPs in bacteria.

Figure 1. A journey toward the biogenesis of a β-barrel outer membrane protein

a. Schematic of the architecture of a generic β-barrel outer membrane proteins (OMP), which consists of a linear arrangement of an even number of antiparallel β-strands (ranging from 8–26) where the first strand and last strand (also referred to as the β-signal) interact at the junction site to form a barrel shape within the membrane. Residues oriented inside the barrel domain (indicated by cyan arrows) are primarily hydrophilic, interacting with solvent, other domains, and/or other proteins, while the residues oriented outside the barrel domain (indicated by red arrows) are almost exclusively hydrophobic, mediating interactions with the hydrophobic core of the membrane. b. An example of an OMP, the structure of Ail (PBD ID 3QRA) from Y. pestis containing eight strands. The first strand is shown in green and the last strand is shown in yellow. c. Gram-negative bacteria contain both an inner membrane and an outer membrane, where the inner membrane partitions the cytoplasm and periplasm and peptidoglycan. The inner membrane contains exclusively α-helical membrane proteins, whereas the outer membrane contains almost exclusively β-barrel outer membrane proteins; only a few examples of the outer membrane containing α-helical membrane proteins are known. d. The biogenesis of an OMP begins within the cytoplasm where it is translated with an N-terminal leader sequence (green). It is then translocated across the inner membrane into the periplasm by the Sec machinery. The periplasmic chaperones SurA (primary pathway) and Skp (rescue pathway) then stabilize and further escort the nascent OMP to the BAM complex for final insertion into the outer membrane. If OMPs become misfolded and cannot be rescued by Skp, they will be degraded by DegP to prevent toxicity to the cell. e. In E. coli, the BAM complex consists of five components: the essential core component BamA, which is an OMP itself, and four accessory lipoproteins termed BamB, BamC, BamD and BamE, each containing an N-terminal post-translational lipid modification that anchors them to the inner leaflet of the outer membrane. Based on microscopy studies, the majority of the soluble domain of BamC is found at the surface of the bacteria, as depicted. However, recent structural studies show close association with BamD, and further studies are required to determine whether the proposed presence of BamC at the surface serves as part of its role within the BAM complex. f. The interaction network within the BAM complex, based on experimental studies, including results from recent structural studies. The BAM complex has been purified as two separate modules containing BamAB and BamCDE.

Figure 2. The structures of the β-barrel assembly machinery complex

a. The individual structures of all the Bam components. BamA (green) contains an N-terminal periplasmic domain consisting of five polypeptide transport-associated (POTRA) repeat domains (P1 – P5) and a C-terminal 16-stranded β-barrel domain. BamB is an eight bladed β-propeller structure containing multiple WD40-like repeats and may serve as a scaffolding protein. BamC, which has been shown to be presented at the surface, contains multiple domains, including an unstructured N-terminal domain followed by two helix-grip domains. BamD contains five tetratricopeptide repeat (TPR) domains and forms the primary interaction with BamA. BamE contains an ααβββ globular fold and interacts with both BamA and BamD, helping to stabilize the intact complex. b,c. The structure of the BAM complex was recently reported with (PDB ID 5D0O and 5AYW) (b) and without BamB (PDB ID 5D0Q and 5EKQ) (c) by X-ray crystallography, revealing an unprecedented 45° shift of the first half of the barrel domain of BamA. The direction of the conformational changes observed within the POTRA domains are indicated. d. More recently, the structure of the BAM complex with BamB was reported using cryoEM (PDB ID 5LJO), revealing a similar conformation as observed in the X-ray crystal structure lacking BamB (part c). e. These structures suggest that the role of the Bam accessory proteins may be to regulate the conformation of BamA during OMP biogenesis. In all structures reported to date, two major conformations have now been observed for BamA. In the first, the barrel domain of BamA shows an ‘inward open’ conformation where the exit pore is occluded, with POTRA5 being located away from the barrel domain to enable access to the lumen from the periplasm. In the second, the barrel domain of BamA shows an ‘outward open’ conformation where the exit pore is now fully open, as POTRA5 shifts to occlude access to the barrel lumen from the periplasm. These conformational states were initially thought to be modulated by BamB, however, both have now been observed in the presence and absence of BamB. Exactly what role the conformational changes serve or how they are regulated remains unknown. The structure of BamA alone (PDB ID 4C4V and 4N75) has also been reported and was found in an ‘inward open’ state; however, BamA alone would presumably be rare in vivo.

Figure 3. Mechanistic models for the role of the BAM complex in OMP biogenesis

a. The first model proposes that β-barrel outer membrane proteins (OMPs) fold intrinsically and just need a locally disturbed membrane to organically fold into. In this model, nascent OMPs are first synthesized in the cytoplasm with an N-terminal leader sequence (removed by proteases at the inner membrane) which directs them to the Sec translocon for transport across in the inner membrane into the periplasm (step 1). Next, the periplasmic chaperones SurA or Skp (not shown) binds the nascent OMPs and delivers them to the BAM complex (step 2), which serves as a catalyst to locally destabilize the membrane bilayer as indicated) and to traffic the nascent OMP into close proximity to the primed membrane for insertion into the outer membrane (step 3). The current hypothesis is that trafficking to the BAM complex may be regulated by the β-signal of the nascent OMP. b. The second model proposes a more systematic mechanism whereby the SurA- and/or Skp-stabilized nascent OMP is delivered to the BAM complex, which then systematically folds it into the membrane. This model shares the initial two steps (step 1 and step 2) with the first model (part a), where nascent OMPs are imported into the periplasm and stabilized by chaperones such as SurA and Skp (not shown). Next, the nascent OMPs are delivered to components of the BAM complex (possibly BamB, BamD, and/or the POTRA domains of BamA) and folding and insertion are initiated by the β-signal of the nascent OMP (step 3)). Upon opening of the lateral gate of BamA, a β-hairpin has been hypothesized to bind the exposed N-terminal strand of BamA by β-augmentation. The barrel of the nascent OMP integrates directly into the barrel of BamA, thus satisfying the requirement that hydrophilic residues point inside the barrel, while hydrophobic residues point outside and mediate interaction with the membrane. The nascent OMP continues to grow either strand by strand or by a β-hairpin at a time (steps 4–6)). To prevent the formation of a super-pore in the outer membrane, the nascent OMP eventually ‘buds’ away from the barrel domain of BamA. We hypothesize that maturation occurs when the first strand of the nascent OMP comes into proximity of the junction site, whereby the last strand of the nascent OMP unpairs from BamA and then pairs with its own first strand (step 7)). This results in the termination of the folding process, forming an independent barrel which then diffuses into the outer membrane, while the BAM complex is left primed for another round of OMP folding.