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 uniq ue beta -chain, all constructed of repeating complement control protein (CC P) modules. The beta -chain, made up of three CCPs, binds tightly to vitami n K-dependent protein S, a cofactor to anticoagulant activated protein C. W hen bound to C4BP, protein S loses its activated protein C cofactor functio n. 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 inte raction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta -chain and were L2T/F45Q, I16SN18S , V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants wer e 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 t o the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.