We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. In one major line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the outer membrane (OM). It was originally proposed that the passenger domain is secreted through a channel formed by the covalently linked beta barrel domain (whence the name autotransporter), but results that we obtained from both biochemical and structural studies are inconsistent with this hypothesis. We found that the insertion of a small linker into the EspP passenger domain effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. By using a site-specific photocrosslinking approach we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues that are trapped in the periplasm (the space between the two cell membranes) interact with the chaperones SurA and Skp. The EspP-BamA interaction is short-lived and can only be detected when passenger domain translocation is stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain prior to its secretion and the Bam complex plays a major role in facilitating both the integration of the beta barrel domain into the OM and the translocation of the passenger domain across the OM. We also found that periplasmic chaperones and specific components of the Bam complex interact with the EspP beta barrel domain in a temporally and spatially regulated fashion. While the chaperone Skp initially interacts with the entire beta barrel domain, BamA, BamB and BamD subsequently interact with discrete beta barrel domain regions. BamB and BamD remain bound to the beta barrel domain longer than BamA and therefore appear to function at a later stage of assembly. Our results suggest that the hitherto enigmatic BamB and BamD proteins play a direct role in the membrane integration of autotransporter beta barrel domains and possibly other beta barrel proteins. Recently, we have also obtained evidence that beta barrel domain begins to fold in the periplasm before it interacts with the Bam complex. In addition to analyzing the assembly of EspP in vivo, we have also reconstituted the assembly of the protein in vitro using purified components. We found that the Bam complex and SurA are both necessary and sufficient to promote the integration of the EspP beta barrel domain into proteoliposomes and the translocation of the passenger domain into the lumen of the vesicles. The reaction does not require ATP or other exogenous energy source. Because passenger domain translocation proceeds in a C-to-N-terminal direction, we were able to examine the assembly of N-terminally truncated EspP derivatives that have different length passenger domains. Interestingly, we found that the rate of assembly is independent of passenger domain length. This observation suggests that the membrane integration of the beta barrel domain is the rate-limiting step in autotransporter assembly. We also found that a single copy of the Bam complex incorporated into nanodiscs is sufficient to catalyze EspP assembly.
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