Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

Autotransporters represent a large family of virulence proteins secreted by Gram-negative bacteria 1-5. All autotransporters contain a cleavable signal peptide, an N-terminal passenger domain that carries the virulence function, and a C-terminal β-domain. Passenger domains vary in size but are usually very large (∼600 - 3000 residues). The β- domains of “classical” autotransporters are typically ∼250 - 300 residues whereas the β-domains of a distinct subfamily of trimeric autotransporters are only ∼70 residues. Autotransporters are translocated across the inner membrane by the Sec machinery. After transiting the periplasm, β-domains are inserted into the outer membrane (OM) as β-barrels and passenger domains are translocated across the OM into the extracellular space. Once translocated, a passenger domain can remain associated with the OM or be cleaved and released from the β-domain 6. The structures of four passenger domains have been solved as either full-length proteins or fragments 7-10 and were shown to contain β-solenoid motifs (see Supplementary Fig. 1 online) 11. The structures of the β-domains of NalP, a classical autotransporter from Neisseria meningitidis 12, and Hia, a trimeric autotransporter from Haemophilus influenzae 13, have also been solved. The NalP β-domain crystallized as a monomeric 12-stranded β-barrel with a ∼30 residue segment of the passenger domain traversing the pore in an α-helical conformation. The Hia β-domain is also a single 12-stranded β-barrrel, but it is assembled from three subunits that each contribute four β-strands. A short passenger domain segment derived from of each of the three subunits is embedded inside the β-domain pore. Several models have been proposed to explain passenger domain translocation 12-15 (recently reviewed by Dautin and Bernstein 16). In one model, the C-terminus of the passenger domain is folded into the β-domain pore in the periplasm in a post-translocation conformation. The prefolded β-domain is then inserted into the OM and the passenger domain is transported across the OM by a concerted mechanism that possibly involves Omp85, an essential protein that promotes OM protein integration and assembly 17. An advantage of this model is that it circumvents the need for one or more passenger domains to be translocated through a relatively small barrel pore in the absence of an external energy source. A second translocation model focuses on the β-solenoid motifs found in passenger domains, which could supply the energy needed for translocation by folding on the extracellular side of the OM once a small portion has reached the cell surface 11. In this model, a short hairpin comprising the C-terminus of the passenger domain is positioned inside the barrel pore with its tip protruding into the extracellular space. Folding at the tip of the hairpin would then pull the rest of the passenger domain through the pore. A third model is based on the observation that the β-domain of IgA protease forms multimeric ring-like structures when the protein is produced in E. coli 18. The central cavity is about 20 A in diameter, and was postulated to transport multiple passenger domains. The focus of this study, EspP, is a classical autotransporter associated with diarrheagenic strains of E. coli 19. It belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporters, whose passengers encode serine proteases that cleave various mammalian proteins 6,20. Biochemical studies have indicated that EspP is a monomer like NalP 15. Once the EspP passenger domain is translocated across the OM, it is cleaved from the membrane embedded β-domain between two asparagine residues (Asn1023/Asn1024) and released from the cell surface. The Asn/Asn cleavage site defines the boundary of the EspP passenger domain (residues 56 -1023) and β-domain (residues 1024 - 1300) 21. Although the passenger domain contains a serine protease motif located at residues 261 - 264, this motif is not used to cleave the two domains 22,23. To learn what happens to the β-domain after cleavage and release of the passenger domain, we determined the crystal structure of the native EspP β-domain at 2.7 A resolution. This is the first structure of an autotransporter β-domain post-cleavage, and it consists of a monomeric 12-stranded β-barrel with its N-terminal 15 residues inserted into the barrel lumen from the periplasmic side. In agreement with a recently proposed autocatalytic cleavage mechanism 24, residues implicated in cleavage are located deep inside the β-barrel, in a region of EspP that would be embedded in the OM. The structure suggests that two discrete conformational changes occur after cleavage and release of the passenger domain: one confers increased stability on the β-domain and another restricts access to the barrel pore. Our structure does not support an oligomeric translocation model, but rather a model in which a single β-barrel facilitates the translocation of a single passenger domain to the extracellular surface 15.