b -domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu 601 -Ala 692 ) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu 601 -Ala 692 in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.

The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "␣-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "␤-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal ␣-domain and a 30-kDa C-terminal ␤-domain. We have previously shown that a recombinant form of the ␤-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31-to 39-amino-acid region found immediately upstream of the ␤-domain that was essential for surface expression. This 31-to 39-amino-acid linker region, together with the ␤-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.

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The essential bacterial membrane protein YidC facilitates insertion and assembly of proteins destined for integration into the inner membrane. It has homologues in both mitochondria and chloroplasts. Here we report the crystal structure of the Escherichia coli YidC major periplasmic domain (YidC EC P1) at 2.5 Å resolution. This domain is present in YidC from Gramnegative bacteria and is more than half the size of the full-length protein. The structure reveals that YidC EC P1 is made up of a large twisted ␤-sandwich protein fold with a C-terminal ␣-helix that packs against one face of the ␤-sandwich. Our structure and sequence analysis reveals that the C-terminal ␣-helix and the ␤-sheet that it lays against are the most conserved regions of the domain. The region corresponding to the C-terminal ␣-helix was previously shown to be important for the protein insertase function of YidC and is conserved in other YidC-like proteins. The structure reveals that a region of YidC that was previously shown to be involved in binding to SecF maps to one edge of the ␤-sandwich. Electrostatic analysis of the molecular surface for this region of YidC reveals a predominantly charged surface and suggests that the SecF-YidC interaction may be electrostatic in nature. Interestingly, YidC EC P1 has significant structural similarity to galactose mutarotase from Lactococcus lactis, suggesting that this domain may have another function besides its role in membrane protein assembly.