Biogenesis of bacterial autotransporter proteins

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We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. In one line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. Using several different biochemical methods we found that the EspP beta domain behaves as a compact monomer and forms a channel that is too narrow to accommodate folded polypeptides. The biochemical data were corroborated by the crystal structure of the beta domain, which we solved in collaboration with Dr. Susan Buchanan and co-workers (NIDDK). Surprisingly, we found that a folded protein domain attached to the N-terminus of EspP is efficiently translocated across the OM and that the native EspP passenger domain folds at least partially in the periplasm. These apparently paradoxical data strongly suggest that an external factor transports the passenger domain across the OM and that the beta domain functions primarily to target the protein to the OM. Our results challenge the prevailing view that the autotransporter beta domain functions as a protein translocase. The crystal structure of the EspP beta domain strongly suggested that a short polypeptide segment that encompasses the beta domain-passenger domain junction resides inside the pore formed by the beta domain following translocation of the passenger domain across the OM. We found, however, that this segment is assembled into the beta domain prior to the initiation of passenger domain translocation. The data strongly suggest that the EspP beta domain and an embedded polypeptide segment are integrated into the OM as a single pre-formed unit. Taken together, our results suggest that the integration of the EspP beta domain into the OM and the translocation of the passenger domain across the OM occur in a single concerted reaction. Interestingly, very recent experiments have suggested that this reaction is mediated by a heterooligomeric complex (Bam complex) that has previously been shown to promote the integration of beta barrel proteins into the bacterial OM.