Biogenesis of bacterial outer membrane proteins

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To obtain insight into OMP biogenesis, we developed a method to trap a modified form of an E. coli autotransporter called EspP stably bound to the Bam complex at a late stage of assembly in vivo. In E. coli, the Bam complex consists of BamA, a protein found in all Gram-negative bacteria that consists of a 16-stranded transmembrane beta barrel domain and five periplasmic polypeptide transport associated (POTRA) domains, and four lipoproteins (BamB-BamE). Using disulfide bond crosslinking, we found that when assembly stalls the C-terminal beta strand of the EspP beta barrel (which contains a highly conserved motif known as the beta signal) forms a rigid interface with the first beta strand of a laterally open form of the BamA beta barrel. In contrast, the N-terminal beta strand of the EspP derivative forms weaker, conformationally heterogeneous interactions with the last two beta strands of BamA that likely represent intermediate assembly states. Recently, we used single-particle cryo-EM to obtain high-resolution structures of the Bam complex bound to the modified form of EspP and to visualize the dynamics of the assembly process. Consistent with the biochemical data, the cryo-EM data show that the first beta strand of the BamA beta barrel forms a stable interaction with the EspP beta signal. The structural data also show that the folding of the EspP beta barrel proceeds via remarkable "hybrid-barrel" assembly intermediates in which membrane integrated beta sheets are attached to BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP assembly intermediates and suggest that a curved beta sheet progressively folds towards BamA to form a barrel-like structure. Along with in vivo experiments that tracked beta barrel folding while the tension of the OM was modified, our results support a model in which the Bam complex harnesses the natural rigidity of the OM to accelerate beta barrel folding. Several antibacterial compounds have been discovered recently that potently inhibit the activity of the essential BamA protein, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors (a natural product called darobactin and two synthetic compounds called Polyphor peptide 7 and MRL-494) on the biogenesis of EspP in vivo. We found that darobactin blocked the binding of the EspP beta signal to BamA, but had no effect on assembly if added at a post-binding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both the binding of the beta signal to BamA and at least one later step of assembly. Taken together with previous studies that analyzed the interaction of darobactin and Polyphor peptide 7 with BamA in vitro, our results strongly suggest that the two compounds inhibit BamA function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the beta signal to BamA initiates the membrane insertion of OMPs. BamA is a member of the Omp85 superfamily of OMPs found in Gram-negative bacteria, mitochondria and chloroplasts that are defined by the presence of a distinctive 16-stranded beta barrel domain and at least one POTRA domain. All of the Omp85 proteins that have been studied to date promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of a largely uncharacterized Omp85 protein family that contains an N-terminal patatin-like (PL) domain as well as a POTRA domain and a C-terminal beta barrel domain. Based in part on the apparent similarity of the PlpD family to another Omp85 protein family, it is widely believed that the PL domain is translocated into the extracellular space (where it functions as a virulence factor) by the covalently linked beta barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and that unlike previously studied Omp85 proteins which are monomeric, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the adjacent beta barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.