The calcium ion and membrane binding structure of the Gla domain of calcium-prothrombin fragment 1

Abstract

The structure of Ca-prothrombin fragment 1 (residues 1-156 prothrombin) has been solved and refined at 2.2-A resolution by X-ray crystallographic methods. The first two-thirds of the Gla domain (residues 1-48) and two carbohydrate chains (approximately 5 kDa) are disordered in crystals of apo-fragment 1. When crystals are grown in the presence of Ca2+ ions, the Gla domain exhibits a well-defined structure binding seven Ca2+ ions, but the carbohydrate is still disordered. Even so, the crystallographic R factor reduced to 0.171. The folding of the Gla domain is dominated by 9-10 turns of three different alpha-helices. These turns produce two internal carboxylate surfaces composed of Gla side chains. A polymeric array of five Ca2+ ions separated by about 4.0 A intercalates between the carboxylate surfaces. The coordination of the Ca2+ ions with Gla carboxylate oxygen atoms and water molecules leads to distorted polyhedral arrangements with mu-oxo bridges in a highly complex array that most likely orchestrates the folding of the domain. The overall mode of interaction of the Ca2+ ions is new and different from any Ca2+ ion-protein interactions heretofore observed or described. The fluorescence quenching event observed upon Ca2+ ion binding is due to a disulfide-pi-electron interaction that causes a 100 degrees reorientation of Trp42 of the Gla domain. The Ca2+ ion interaction also affords the N-terminus protection from acetylation because the latter is buried in the folded structure and makes hydrogen-bonding salt bridges with Gla17, Gla21, and Gla27. The Gla domain and its trailing disulfide unit associate intimately and together give rise to a domain-like structure. Electrostatic potential calculations indicate that the Gla domain is very electronegative. Since most of the carboxylate oxygen atoms of Gla residues are involved in Ca2+ ion binding, leaving only a few for bridging Ca2+ ion-phospholipid interactions, the role of bridging Ca2+ ions might be generally unspecific, with Ca2+ ions simply intervening between the negative Gla domain and negative head groups of the membrane surface. The folding of the kringle structure in apo- and Ca-fragment 1 is essentially the same. However, the Ser36-Ala47 helix of the Gla domain pivots around Cys48, shifting by approximately 30 degrees, and the helix encroaches on the kringle producing some concomitant changes. These might be related to the protection of carbohydrate carrying Asn101 from acetylation in the Ca-fragment 1 structure.

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

“…A structural model of the human prothrombin Gla domain was constructed by using the x-ray structure of bovine prothrombin as template, 39 with Accelrys (San Diego, CA) and ICM (MolSoft, San Diego, CA) software packages running on a Silicon Graphics Fuel (Mountain View, CA) workstation or a Dell (Round Rock, TX) Linux personal computer. The model of the human PS Gla-TSR-EGF1 modules has been published elsewhere.…”

“…Thus, we decided to replace the face 1 and face 2 residues of Gla FII -PS. In this mutant, designated F12 PS -Gla FII -PS, residues 21,23,24,28,33,34,35,36,39,41, and 45 were replaced by the corresponding residues of PS ( Figure 1). This mutant comprised an Ala residue of prothrombin origin at position 42, disrupting the integrity of the aromatic amino acid stack Pro35-Pro42 loop that is potentially important for PS functions.…”

Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

“…The N-terminal domain, encoded on a separate exon, contains the first 37 amino acid residues including all 10 y-carboxyglutamic acid (Gla) residues [2]. This so-called Gla domain binds seven Ca r+ ions [3,4], an event mediated by the Gla residues, and thereby attains a membrane-interactive ability through the exposure of hydrophobic side chains [4,5]. Studies of the roles of the individual Gla residues in factor IX [6], protein C [7][8][9][10] and prothrombin [11], all of which are homologous to fVII, have been conducted.…”

Factor VIIa and the extracellular domains of human tissue factor form a compact complex: A study by X‐ray and neutron solution scattering

“…The NMR structures of the EGF-1 domain of human factors IX and X [6,7] provide a close model for the EGF-I domain in FVIIa. Finally the known crystal structure of the N-terminal Gla domain of prothrombin [8] may be used as a model for that in FVIIa. While an atomic model for FVIIa can be proposed, there is no information on the relative spatial arrangement of its four domains.…”