Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF-2 domain of blood coagulation factor VII

The cyclic dodecapeptide, disulfide-cyclo-[H-Cys-Val-Asn-C lu-Asn-Gly-G ly- Cys(Acm)-Glu-Gln-Tyr-Cys-OH], which corresponds to the 91-102 sequence of t he second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid-phase procedures. It was shown to be an in hibitor at the key step in the induction of coagulation by the extrinsic pa thway, i.e. the factor VII/tissue factor-catalyzed activation of coagulatio n factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer-modeled via molecular mechanics. Structures w ere calculated based on 112 distance and nine dihedral angle constraints. T he resulting backbone structures were classified into two structural subset s: one which exhibited a twisted '8'-shaped folding and another describing an open, circular 'O' outline. The local backbone structures of segments As n(3)-Glu(4)-Asn(5), Gly(7)-Cys(8) and Gln(10)-Tyr(11) were well preserved a mong the two subsets. Apart from the unrestrained N- and C-termini, Gly(6) and Glu(9) sites exhibited marked local disorder between the two subsets, s uggesting localized flexible hinges likely to govern tertiary structure int erconversion between the two subsets. Two transient hydrogen bonds were ide ntified from pH chemical shift titrations by matching the pK(a) values of N H and carboxylate groups, which supported the occurrence of the '8' structu re, and agreed with temperature coefficients of peptidyl NH resonances. Str ucture-function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide-bonded loop in factor VII w hich the peptide represents.