Mutational analysis of the substrate binding site of human complement factor D.
Complement factor D is a serine protease with a single natural substrate, C3b-complexed factor B, and very low catalytic activity against synthetic esters. The recently solved X-ray crystal structure of factor D has demonstrated certain key differences from other serine protease in the conformation of residues of the catalytic triad and the substrate-binding regions. To investigate possible contributions of unique amino acid substitutions to these distinct structural and functional features of factor D, we constructed a series of mutants by substituting trypsin substrate-binding residues for the corresponding factor D residues. Wild-type and seven mutant factor D cDNAs were expressed stably in Chinese hamster ovary cells, and the recombinant proteins were purified from culture supernatants and assayed by hemolytic, proteolytic, and esterolytic assays. The combined results indicate that residues Thr-198, Ser-199, Arg-202, and perhaps also Val-203 provide determinants for substrate binding and catalysis. The data also provide additional support for the hypothesis that the proteolytically active conformation of the active center of factor D is induced by its substrate, C3bB.
Kim
S
S
Department of Microbiology, University of Alabama at Birmingham 35294.
Narayana
S V
SV
Volanakis
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D011415:Complement Factor B / Q000378:metabolism; D011416:Complement Factor D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry
D011994:Recombinant Proteins; D014357:Trypsin; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B