Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein.
C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.
Webb
J H
JH
Division of Clinical Chemistry, Department of Laboratory Medicine, Lund University, University Hospital Malmö, Malmö S-205 02, Sweden.
Villoutreix
B O
BO
Dahlback
B
B
Blom
A M
AM
D001483:Base Sequence; D001665:Binding Sites; D001779:Blood Coagulation Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D011951:Receptors, Complement / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001779:Blood Coagulation Factors; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D011951:Receptors, Complement; D011994:Recombinant Proteins