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 Ca(2+)-binding sites in a region comprising four EGF modules. The EGF 3-4 module pair constitutes the smallest fragment that retains one high-affinity Ca(2+)-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca(2+) binding compared to other EGF-containing proteins characterized so far. Extensive chemical shift effects caused by Ca(2+) binding to the EGF 3-4 module pair are observed, particularly from Ca(2+) binding to the high-affinity site in EGF 4. Ca(2+) binding 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, respectively. 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 similar Ca(2+) affinities and backbone dynamics. Further, broadening of (1)H resonances involving residues in the major beta-sheet of EGF 3 and (15)N exchange terms, primarily in the N-terminal part of the protein, indicate the presence of slow exchange on a microsecond to millisecond time scale. (15)N spin relaxation data suggest that the module pair has a well-defined relative orientation between EGF modules 3 and 4 and has a significantly anisotropic rotational diffusion tensor in solution.