Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein

Anticoagulant protein S interacts with the complement regulatory protein C4 b-binding protein (C4BP) via its sex-hormone-binding globulin (SHBG)-like r egion, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4B P or to have any anticoagulant activity. To determine the relative importan ce of the two G domains in protein S for C4BP protein binding, three recomb inant protein S chimeras were produced having either of the two globular do mains, or the whole SHB6-like globulin region, replaced by corresponding pa rts from Gas6. The chimeras were tested for binding to immobilized C4BP usi ng surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C 4BP of both chimeras containing individual G domains from protein S, were l ower than that of wild-type protein S, Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, w hich had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly wit h C4BP. Our results demonstrate that both G domains of protein S an involve d in the interaction between protein S and C4BP and that full affinity bind ing is dependent on contributions from both domains.