The EGF-like Modules of Anticoagulant Protein S. Studies of Ca2+ binding and module interactions

Abstract: Protein S functions as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. In plasma protein S circulates in two forms; 30-40 % circulates as free protein S molecules while the remaining 60-70 % exists in a 1:1 complex with C4b-binding protein (C4BP). Only the free form of protein S functions as a cofactor to APC. Protein S is a mosaic protein composed of four discrete modules; a g-carboxyglutamic acid (Gla)-containing module, a thrombin-sensitive module (TSR), four epidermal growth factor (EGF)-like modules and a C-terminal sex hormone binding globulin (SHBG)-like module. The Gla-module contains 11 Gla residues, which require vitamin K for their biosynthesis, and anchors the protein to the membrane. All four EGF-like modules contain another modified amino acid residue, erythro-b-hydroxyaspartic acid (Hya) or erythro-b-hyroxyasparagine (Hyn). The three C-terminal modules also contain a concensus sequence indicating Ca2+ binding. The C-terminal SHBG-like module contains the binding site for C4BP. The aim of this work has been to study the Ca2+ binding affinity of the EGF-like modules, and the involvement of these modules in interactions with other proteins. Fragments containing one, two, three or four EGF-like modules have been isolated. This includes the chemically synthesized single EGF modules 3 and 4. These two modules were also expressed as a pair in a procaryotic system and folded to a native conformation. EGF 1-4, EGF 1-3, EGF 2-3 and EGF 2-4 were expressed in a baculovirus based insect cell system. The Ca2+ affinity in the modules was determined either by NMR monitored titrations or using chromophoric chelators. We found that the single modules EGF 3 and 4 bound Ca2+ in the low mM range (Kd 6-9 mM). In the EGF 3-4 pair the N-terminal site binds Ca2+ with an affinity that is almost identical to that of free module 3, whereas the affinity of the C-terminal module is greatly enhanced. Compared to the single module the affinity is ~8600-fold higher for EGF 4 in the pair. It thus appears as if the major effect on Ca2+ binding is excerted by the module N-terminal of the Ca2+ binding one. EGF modules 1-4 and 2-4 each was found to contain two very high affinity Ca2+ binding sites; with Kd from 10-8 to 10-6 M, at physiological ionic strength. In contrast, the Ca2+ affinities of EGF 2-3 and EGF 1-3 is two to four orders of magnitude lower. These data imply that also modules C-terminal of the Ca2+ binding one influence the Ca2+ affinity. By using the recombinant EGF modules as inhibitors in a clotting assay, it was shown that the interaction between protein S and APC was mediated by recombinant fragments which contained the first EGF module (EGF 1-4 and EGF 1-3). Yet, EGF 1-4 had an almost 10-fold stronger inhibitory effect than EGF 1-3 for reasons that not yet understood. Using a solid-phase system a direct interaction between protein S and factor Xa was also observed. The Ca2+ binding studies, as well as the experiments where the interactions of protein S were studied indicated that module-module interactions are crucial to the biological function of protein S.