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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 outer-membrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7-[Angstrom] structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a [beta]-helix, with three extrahelical motifs, [beta]-hairpin, a four-stranded [beta]-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 [beta]-helical motifs that form the central domain of the adhesin, because it is itself a [beta]-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.

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 [degrees] C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 [Angstrom] and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kda) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.