In recent experiments we have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. We have been examining the mechanism by which the EspP passenger domain is translocated across the OM. Using Blue Native PAGE, analytical ultracentrifugation and other 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 of the autotransporter beta domain as a protein translocase. We have also obtained insights 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 very recently supported by the crystal structure of the EspP beta domain, which we solved in collaboration with the Buchanan laboratory (LMB, NIDDK). The structure showed 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.
| Ieva, Raffaele; Skillman, Kristen M; Bernstein, Harris D (2008) Incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter. Mol Microbiol 67:188-201 |
| Barnard, Travis J; Dautin, Nathalie; Lukacik, Petra et al. (2007) Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat Struct Mol Biol 14:1214-20 |
| Dautin, Nathalie; Bernstein, Harris D (2007) Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol 61:89-112 |
| 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 |
