The process of translocating a large passenger domain by a much smaller beta-domain is currently not understood. Described below are three models that have been proposed to explain passenger domain translocation. In one model, the C-terminus of the passenger domain is folded into the beta-domain pore in the periplasm in a post-translocation conformation. The prefolded beta-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. 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 unusual architecture of passenger domains, which all appear to contain beta-solenoid motifs. These motifs 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. 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 beta-domain of IgA protease forms multimeric ring-like structures when the protein is produced in E. coli. The central cavity is about 20 A in diameter, and was postulated to transport multiple passenger domains. This model is considered unlikely for the majority of autotransporters. A major focus of this project is EspP, a classical autotransporter associated with diarrheagenic strains of E. coli. It belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporters, whose passengers encode serine proteases that cleave various mammalian proteins. Biochemical studies have indicated that EspP is a monomer. Once the EspP passenger domain is translocated across the OM, it is cleaved from the membrane embedded beta-domain between two asparagine residues (N1023/N1024) and released from the cell surface. The Asn/Asn cleavage site defines the boundary of the EspP passenger domain (residues 56-1023) and beta-domain (residues 1024 1300). Although the passenger domain contains a serine protease motif located at residues 261-264, this motif is not used to cleave the two domains. Our goals for this project are to solve crystal structures of the pre- and post-cleavage forms of one or more autotransporters and to design experiments to probe substrate translocation across the outer membrane. Structure determination of a bacterial autotransporter To learn what happens to the &#946;-domain after cleavage and release of the passenger domain, we determined the crystal structure of the native &#946;-domain of EspP at 2.7 resolution in 2007. This is the first structure of an autotransporter &#946;-domain post-cleavage, and it consists of a monomeric 12-stranded &#946;-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, residues implicated in cleavage are located deep inside the &#946;-barrel, in a region of EspP that would be embedded in the OM. Interestingly, the structure suggests that two discrete conformational changes occur after cleavage and release of the passenger domain, one that confers increased stability on the &#946;-domain and another that restricts access to the barrel pore. Our structure does not support an oligomeric translocation model, but rather a model in which a single &#946;-barrel facilitates the translocation of a single passenger domain to the extracellular surface. During the past two years, we have been working on the structure of EspP in its pre-cleavage conformation. Several mutants whose passenger domains are translocated to the extracellular space but are not cleaved have been cloned and expressed. By varying lengths of passenger domain in these constructs, we optimized crystallization conditions and have recently collected high resolution diffraction data on three EspP mutants that translocate the passenger but do not cleave it. We are currently building models into the electron density, but already it is clear that some aspects of the proposed cleavage mechanism will be revised through this work. We aim to solve another two or three mutant structures in the coming months and publish a comprehensive analysis of the pre-cleavage state, including cleavage mechanism and passenger release, in 2010. A pre-cleavage structure will also allow us to attempt structure-based mutagenesis to test between the proposed mechanisms of passenger translocation. As of August 2010, all four mutant structures have been fully refined and we are currently writing a manuscript for publication. The new structures give us additional insight into the cleavage mechanism, but we still do not know exactly how the passenger domain is tranlocated. To learn more about protein translocation, we initiated a new autotransporter project which focuses on proteins related to EspP but having a different gene organization. Whether these proteins have the same structure and function the same way, is currently unclear. To date we have 3.5 data on one such protein and we are working to improve the resolution and obtain phases for structure solution.