Localization of a Hydrophobic Binding Site for Anticoagulant Protein S on the β-Chain of Complement Regulator C4b-binding Protein

Abstract

C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven α-chains and one unique β-chain, all constructed of repeating complement control protein (CCP) modules. The β-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 β-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the β-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 Ile16, Val 18, Val31, and Ile33 as crucial for protein S binding, with secondary effects from Leu38 and Val39. In addition, Lys41 and Lys42 contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.