Proton NMR assignment and secondary structure of the calcium-free form of the amino-terminal epidermal growth factor like domain in coagulation factor X

Abstract

Blood coagulation factor X is composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). The N-terminal EGF like domain in factor X (fX-EGFN), residues 45-86 of the intact protein, contains a .beta.-hydroxylated aspartic acid and has one Ca2+-binding site. Using 2D NMR techniques, we have made a full assignment of the 500-MHz 1H NMR spectrum of Ca2+-free fX-EGFN. On the basis of this assignment and complementary NOESY experiments, we have also determined the secondary structure of Ca2+-free fX-EGFN in water solution. Residues 45-49 are comparatively mobile, whereas residues 50-56 are constrained by two disulfide bonds to one side of an antiparallel .beta.-sheet involving residues 59-64 and 67-72. Another antiparallel .beta.-sheet involves residues 76-77 and 83-84. A small, parallel .beta.-sheet connects residues 80-81 and 55-56 and thereby orients the two antiparallel .beta.-sheets relative to each other. Four .beta.-turns are identified, involving residues 50-53, 56-59, 64-67, and 73-76. Residues 78-82 adopt an extended bend structure. On the basis of secondary structure and the location of the three disulfide bonds, we find that Asp 46, Asp 48, and Hya 63 are sufficiently close to each other to form a Ca2+-binding site. However, the amino terminus of the Ca2+-free form of fX-EGFN is not part of a triple-stranded .beta.-sheet as in other EGF like peptides. Differences and similarities between fX-EGFN and murine EGF with respect to secondary structure and conformational shifts are discussed.