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.
Muranyi
A
A
Physical Chemistry 2, Lund University, P.O. Box 124, S-221 00 Lund, Sweden and Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden. andreas.muranyi@fkem2.lth.se
Evenäs
J
J
Stenberg
Y
Y
Stenflo
J
J
Drakenberg
T
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016880:Anisotropy; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D017293:Protein S; D004815:Epidermal Growth Factor; D002118:Calcium