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. 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. In a second line of investigation we have obtained insight into the mechanism by which the EspP passenger domain is cleaved from the cell surface. We found that the EspP passenger domain is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor undergoes proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp1120) and an asparagine (Asn1023) at the P1 position of the cleavage junction are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp1120 and Asn1023 form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids. Our proposed cleavage mechanism was supported by the crystal structure of the EspP beta domain, which we solved in collaboration with Dr. Susan Buchanan and her colleagues (LMB, NIDDK). The structure shows that the beta domain forms a 12-stranded beta barrel with the passenger / beta domain cleavage junction located inside the barrel pore, approximately mid-way between the extracellular and periplasmic surfaces of the OM. In very recent studies we showed that the polypeptide segment that encompasses the cleavage junction is assembled into the beta barrel prior to the translocation of the passenger domain across the OM. 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 raise the possibility 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.
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| Bernstein, Harris D (2007) Are bacterial 'autotransporters'really transporters? Trends Microbiol 15:441-7 |
| Dautin, Nathalie; Barnard, Travis J; Anderson, D Eric et al. (2007) Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism. EMBO J 26:1942-52 |
