EGF-like module pair 3-4 in vitamin K-dependent protein S: Modulation of calcium affinity of module 4 by module 3, and interaction with factor X

Calcium-binding epidermal growth factor (EGF)-like modules are found in num erous extracellular and membrane proteins involved in such diverse processe s as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the antico agulant enzyme activated protein C, has four EGF-like modules in tandem wit h the three C-terminal modules each harbouring a Ca2+-binding consensus seq uence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca 2+-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca2+ affinity of i ndividual modules have been identified. As a step towards an analysis of th e structural basis of the high Ca2+ affinity, we expressed the Ca2+-binding EGF pair 3-4 from human protein S. Correct folding was shown by H-1 NMR sp ectroscopy. Calcium-binding properties of the C-terminal module were determ ined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by H-1-N-15 NMR spectroscopy. At physiologica l pH and ionic strength, the dissociation constants for Ca2+ binding were 1 .0 x 10(-6) M and 4.8 x 10(-3) M for modules 4 and 3, respectively, i.e. th e calcium affinity of the C-terminal site was about 5000-fold higher than t hat of the N-terminal site. Moreover, the Ca2+ affinity of EGF 4, in the pa ir 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF mo dules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity. (C) 1999 Aca demic Press.