title: Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.
abstract: Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.
authors.affiliation: Division of Clinical Immunology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: A
authors.initials: A
authors.lastname: Circolo
authors.forename: H
authors.initials: H
authors.lastname: Jing
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: S V
authors.initials: SV
authors.lastname: Narayana
authors.forename: J E
authors.initials: JE
authors.lastname: Volanakis
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D020134:Catalytic Domain; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D011415:Complement Factor B / Q000235:genetics / Q000378:metabolism*; D011416:Complement Factor D / Q000378:metabolism; D004546:Elapid Venoms / Q000378:metabolism; D006461:Hemolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004546:Elapid Venoms; C016310:cobra venom factor; D001224:Aspartic Acid; D001216:Asparagine; D050577:Complement C3-C5 Convertases; D011416:Complement Factor D; D011415:Complement Factor B