H-1, N-15 and C-13 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 Ca2+-binding sites with unusually high affinity, The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (E GF) modules of protein S is the smallest fragment that retains high-affinit y Ca2+ binding and is therefore useful for investigating the structural bas is of this property. Heteronuclear multidimensional nuclear magnetic resona nce experiments were used to obtain extensive assignments of the H-1,N-15 a nd C-13 resonances of the module pair with one Ca2+ bound in EGF 4, In addi tion, nearly complete assignments or the H-1 resonances of the isolated Ca2 +-free EGF 3 module were obtained, The assignment process was complicated b y broadening of several resonances, spectral heterogeneity caused by cis-tr ans isomerisation of the peptide bond preceding Pro-168, and dimerisation, Analysis of weighted average secondary chemical shifts, (3)J(HNH alpha) cou pling constants, and NOE connectivities suggest that both EGF modules in th is fragment adhere to the classical secondary structure of EGF modules, con sisting of one major and one minor anti-parallel beta-sheet, (C) 2000 Feder ation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.