Characterization of the EGF-like module pair 3-4 from vitamin K-dependent protein S using NMR spectroscopy reveals dynamics on three separate time scales and extensive effects from calcium binding

Protein S, a cofactor of anticoagulant activated protein C, exhibits three high-affinity Ca2+-binding sites in a region comprising four EGF modules. T he EGF 3-4 module pair constitutes the smallest fragment that retains one h igh-affinity Ca2+-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca2+ binding compared to other EGF-containing proteins characterized so far. Extensive chemical s hift effects caused by Ca2+ binding to the EGF 3-4 module pair are observed , particularly from Ca2+ binding to the high-affinity site in EGF 4. Ca2+ b inding to the high-affinity site in EGF 4 and the low-affinity site in EGF 3 is associated with slow and fast exchange on the NMR time-scale, respecti vely. We show the presence of two isoforms, characterized by a cis or trans Lys 167-Pro 168 peptide bond, that do not convert on time scales that were accessible to the experiments (k(ex) < 0.2 s(-1)). Both conformers have si milar Ca2+ affinities and backbone dynamics. Further, broadening of H-1 res onances involving residues in the major <beta>-sheet of EGF 3 and N-15 exch ange terms, primarily in the N-terminal part of the protein, indicate the p resence of slow exchange on a microsecond to millisecond time scale. N-15 s pin relaxation data suggest that the module pair has a well-defined relativ e orientation between EGF modules 3 and 4 and has a significantly anisotrop ic rotational diffusion tensor in solution.