1H, 15N and (13)C assignments and secondary structure of the EGF-like module pair 3-4 from vitamin K-dependent protein S.
Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.
Muranyi
A
A
Physical Chemistry, Lund University, Sweden. andreas.muranyi@fkem2.lth.se
Evenäs
J
J
Stenberg
Y
Y
Stenflo
J
J
Drakenberg
T
T
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002247:Carbon Isotopes; D004815:Epidermal Growth Factor / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D017293:Protein S / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D017293:Protein S; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D006859:Hydrogen; D002118:Calcium