Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica

The giant non-fimbrial adhesin siie of salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. siie is a 595 kda protein composed of 53 repetitive bacterial immunoglobulin (big) domains and the only known substrate of the spi4-encoded type 1 secretion system (t1ss). the crystal structure of big50-52 of siie revealed two distinct ca2+-binding sites per big domain formed by conserved aspartate or glutamate residues. in a mutational analysis ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the big domains of siie. amounts of secreted siie diminish with a decreasing number of intact ca2+-binding sites. big domains of siie contain distinct ca2+-binding sites,with type i sites being similar to other t1ss-secreted proteins and type ii sites newly identified in siie. we functionally and structurally dissected the roles of type i and type ii ca2+-binding sites in siie,as well as the importance of ca2+-binding sites in various positions of siie. type i ca2+-binding sites were critical for efficient secretion of siie and a decreasing number of type i sites correlated with reduced secretion. type ii sites were less important for secretion,stability and surface expression of siie,however integrity of type ii sites in the c-terminal portion was required for the function of siie in mediating adhesion and invasion. © 2017 peters et al.