Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein
Författare
Summary, in English
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.
Avdelning/ar
Publiceringsår
2001
Språk
Engelska
Sidor
4330-4337
Publikation/Tidskrift/Serie
Journal of Biological Chemistry
Volym
276
Issue
6
Dokumenttyp
Artikel i tidskrift
Förlag
American Society for Biochemistry and Molecular Biology
Ämne
- Other Basic Medicine
- Medicinal Chemistry
Status
Published
Forskningsgrupp
- Clinical Chemistry, Malmö
- Protein Chemistry, Malmö
ISBN/ISSN/Övrigt
- ISSN: 1083-351X