A 76-AMINO ACID DISULFIDE LOOP IN THE YERSINIA-PSEUDOTUBERCULOSIS INVASIN PROTEIN IS REQUIRED FOR INTEGRIN RECEPTOR RECOGNITION

The Yersinia pseudotuberculosis invasin protein is a 986-amino acid pr otein that promotes bacterial penetration into mammalian cells by avid ly binding multiple beta1-chain integrins. A 192-amino acid carboxyl-t erminal domain of invasin was previously shown to be sufficient for bi nding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-medi ated cell binding and entry. Bacterial mutants that were altered at ei ther of 2 cysteine residues in the binding domain of invasin were comp letely defective for entry. Purified invasin protein derivatives alter ed at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of prote olytic products of invasin in the presence or absence of reducing agen t provided evidence of an intrachain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [H-3]i odoacetate indicated that these 2 cysteines were normally disulfide-bo nded. A treatment that resulted in the maximal reduction of the disulf ide bond also resulted in maximal loss of cell attachment activity. Th ese results indicate that the 76-amino acid disulfide loop at the carb oxyl terminus of invasin is required for recognition by integrins.