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 protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.
Department of Medicine, Tufts-New England Medical Center Hospital, Boston, Massachusetts 02111.
J M
JM
Leong
P E
PE
Morrissey
R R
RR
Isberg
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D015011:Yersinia pseudotuberculosis / Q000235:genetics / Q000502:physiology*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D003062:Codon; D004220:Disulfides; D016023:Integrins; D007461:Iodoacetates; D011994:Recombinant Proteins; C053476:invasin, Yersinia; D004229:Dithiothreitol; D019807:Iodoacetic Acid