Type II protein secretion in gram-negative pathogenic bacteria: The study of the structure/secretion relationships of the cellulase CeI5 (formerly EGZ) from Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulas e Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out ). Cel5 is composed of two domains, a large N-terminal catalytic domain (39 0 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagen esis and structural analysis permitted us to investigate the structure/secr etion relationships with respect to the catalytic domain of Cel5. The 3D st ructure of the catalytic domain was solved by molecular replacement at 2.3 Angstrom resolution. Cel5 exhibits the (beta/alpha)(8) structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-media ted suppression allowed us to predict positions of importance in the molecu le in relation to structure and catalysis. Remarkably, all of the predictio ns proved to be correct when compared with the present structural informati on. Mutations of Arg57, which is located at the heart of the catalytic doma in, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remar kably strong constraints upon folding. In particular, an Arg-to-His mutatio n yielded a species that folded to a stable conformation close to, but dist inct from the wild-type, which however was not secretable. We discuss the r elationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respe ctive secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exopr oteins. (C) 2001 Academic Press.