Crystallographic Studies of Invasin, a Bacterial Adhesion Molecule from Yersinia pseudotuberculosis

Bacterial pathogens, such as Yersinia pseudotuberculosis, must bind and enter normally non-phagocytic cells to establish infection. The protein responsible for mediating uptake of the bacterium is a 986-residue outer membrane protein called invasin. Invasin binds to several members of the beta 1 integrin family, presumably activating a reorganization of the host cytoskeleton to form pseudopods that envelop the bacterium. Integrin binding has been localized to the extracellular region of invasin (Inv497) comprised by the COOH-terminal 497 residues. In order to gain insight into host cell entry by Yersinia pseudotuberculosis, we solved the 2.3 Šcrystal structure of Inv497. The structure reveals five domains that form a 180 Šrod with structural similarities to tandem fibronectin-III domains. The integrin-binding surfaces of invasin and fibronectin include similarly located key residues, but in the context of different folds and surface shapes. The structures of invasin and fibronectin provide an example of convergent evolution, in which invasin presents an optimized surface for integrin binding compared with host substrates. We have also initiated structural analyses of the NH2-terminal ~500 residues of invasin, which are required for outer membrane localization and for presentation of the integrin-binding region of invasin. We expressed this region of invasin as inclusion bodies in E. coli, and refolded the protein in the presence of detergents. We have also obtained microcrystals of this membrane protein. Circular dichroism studies indicate that this region of invasin is composed of mainly beta-structure. As the transmembrane regions of outer membrane proteins of known structure are beta-barrels, this region of invasin is also presumed to fold into such a structure. Proteolysis experiments suggest that the NH2-terminal 70 amino acids of invasin may form a separate domain from the invasin transmembrane region, analogous to that found in another outer membrane protein, the sucrose-specific porin ScrY. Equilibrium sedimentation analytical ultracentrifugation studies indicate the protein is monomeric in solution. Black bilayer experiments suggest that this region of the protein does not contain a pore and thus plays the role of an outer membrane anchor for the presentation of the integrin-binding domain on the cell surfac