Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor.
Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.
University of Washington, Department of Microbiology, Box 357242, Seattle, WA 98195-7242, USA.
Cristina P
CP
Van Loy
Evgeni V
EV
Sokurenko
Ram
R
Samudrala
Steve L
SL
Moseley
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018830:Adhesins, Escherichia coli; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D018958:CD55 Antigens / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism; D002701:Chloramphenicol / Q000494:pharmacology; D024141:Collagen Type IV / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004847:Epithelial Cells / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D001743:Urinary Bladder / Q000166:cytology
D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; D000596:Amino Acids; D000900:Anti-Bacterial Agents; D018958:CD55 Antigens; D024141:Collagen Type IV; C465564:Dr adhesin, E coli; C465563:DraE protein, E coli; D029968:Escherichia coli Proteins; D006388:Hemagglutinins; D021122:Protein Subunits; D002701:Chloramphenicol