Biogenesis of bacterial outer membrane proteins

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

To obtain insight into OMP biogenesis, we developed a method to trap a modified form of an E. coli O157:H7 autotransporter called EspP stably bound to the Bam complex at a late stage of assembly in vivo. 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 beta barrel of BamA, the central subunit of the Bam complex. 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 OM tension was modified, our results support a model in which the Bam complex harnesses OM elasticity to accelerate beta barrel folding. Recently, we also revisited a surprising observation reported over 20 years ago that an eight-stranded E. coli beta barrel protein called OmpA can be assembled into a native structure in vivo when it is expressed as two non-covalently linked fragments. After introducing single cysteine residues into the two fragments we found that disulfide bonds between beta strands 4 in the N-terminal fragment and 5 in the C-terminal fragment form in the periplasmic space and greatly increase the efficiency of assembly of split OmpA, but only if the cysteine residues are engineered in perfect register. In contrast, we observed only weak disulfide bonding between beta strands 1 in the N-terminal fragment and 8 in the C-terminal fragment that would form a closed or circularly permutated beta barrel. Our results not only demonstrate that beta barrels begin to fold into a beta sheet-like structure before they are integrated into the OM, but also help to discriminate between different models of OMP biogenesis. Several years ago we showed that the assembly of small, monomeric OMPs can be reconstituted in vitro using only proteoliposomes containing the purified Bam complex and a molecular chaperone (SurA). Recently, we showed that we could extend the utility of our assay by reconstituting the assembly of a more complex OMP, the trimeric porin OmpC. Trimeric porins are among the most abundant OMPs in E. coli, but their assembly is poorly understood. We found that in vitro synthesized OmpC was inserted into proteoliposomes that contained the Bam complex and folded into heat-stable trimers by passing through a short-lived dimeric intermediate. Interestingly, OmpC assembly was also dependent on the addition of lipopolysaccharide (LPS), a glycolipid located exclusively in the OM. Our results strongly suggest that trimeric porins form through a stepwise process that requires integration of monomers into the OM in an assembly-competent state. Furthermore, our results provide surprising evidence that an interaction with LPS is required not only for trimerization, but also for the productive insertion of monomers into the lipid bilayer.