Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cough agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria. FHA is secreted by means of the two-partner secretion (TPS) pathway. Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion. A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outermembrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7-Å structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a ␤-helix, with three extrahelical motifs, a ␤-hairpin, a fourstranded ␤-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain. The structure thus explains why the TPS domain is able to initiate folding of the ␤-helical motifs that form the central domain of the adhesin, because it is itself a ␤-helical scaffold. It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter. This structure is representative of the TPS domains found so far in >100 secreted proteins from pathogenic bacteria. It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

SummaryThe sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/ FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a ␤-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.Targeting and translocation of soluble and integral membrane proteins to the ad hoc subcellular compartments are essential for cell function and organelle biogenesis. Transport of proteins across membranes and their insertion into membranes are typically mediated by proteinaceous transmembrane complexes, and some of these pathways have been conserved throughout evolution (1). With their complex envelope structure, Gram-negative bacteria and eukaryotic organelles face similar challenges for the translocation of proteins (2-4). In the outer membrane of Gram-negative bacteria, specific membrane-embedded ␤-barrel proteins are essential components of protein transport machineries (5). Similarly, polypeptide-transporting ␤-barrel proteins are also found in the outer membranes of eukaryotic organelles of prokaryotic origin such as chloroplasts and mitochondria (6, 7).The recently revealed Omp85/TpsB superfamily of outer membrane proteins is dedicated to protein transport in most major kingdoms of life, although no members have been identified in Archaebacteria yet (8,9). TpsB transporters are found in two-partner secretion systems, developed by Gram-negative bacteria for the secretion of large "TpsA" proteins destined to the cell surface or the milieu and serving mostly as virulence factors (10). The other members of the Omp85/TpsB superfamily are transporter proteins included in large hetero-oligomeric complexes, such as the Toc75 homologs in chloroplasts, the Tob55/ Sam50 homologs in mitochondria and the Omp85 homologs in Gramnegative bacteria. These transporters mediate inward protein transport across the chloroplast outer membrane and assembly of ␤-barrel proteins into the outer membrane of mitochondria and Gram-negative bacteria, respectively (11-15).Although their sizes and origins vary, the proteins of the TpsB/ Omp85 superfamily are phylogenetically related and have been postulated to derive from a common ancestor, probably a simple prokaryotic cha...