Biogenesis of bacterial outer membrane proteins

National Institute Of Diabetes And Digestive And Kidney Diseases

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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 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 lipid facing surface of the last two beta strands of BamA that likely represent intermediate assembly states. The results indicate that BamA forms a hybrid barrel with client proteins during their assembly. Based on our results, we proposed that BamA catalyzes the membrane insertion of partially folded beta barrels by a novel 'swing' mechanism. We are currently testing our model by determining the structure of the modified form of EspP bound to the Bam complex. Using the same EspP derivative, we also recently obtained conclusive evidence that the passenger domain of autotransporters traverses the OM through the lumen of the BamA beta barrel. Several years ago we reconstituted OMP assembly in vitro, but the reactions originally involved the use of urea-denatured protein purified from inclusion bodies. We subsequently found that the E. coli Bam complex also catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly than their urea-denatured counterparts. 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. Many OMPs contain a conserved C-terminal 'beta signal' motif, but the function of this sequence is unclear. We found that beta signal mutations slightly delayed the assembly of several model E. coli OMPs in vivo by reducing their affinity for the Bam complex and led to their partial degradation in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of the periplasmic chaperone Skp suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the beta signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions.