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, Val¹⁸, Val³¹, and Ile³³ as crucial for protein S binding, with secondary effects from Leu³⁸ and Val³⁹. In addition, Lys⁴¹ and Lys⁴²contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.