Abstract
Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg(2+) ions and four Ca(2+) ions in the GLA domain, one Ca(2+) ion in the EGF1 domain and one Ca(2+) ion in the protease domain. Further, FVIIa contains an Na(+) site in the protease domain. Since Na(+) and water share the same number of electrons, Na(+) sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na(+) site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg(2+), Ca(2+) and Rb(+) ions. In this structure, Rb(+) replaced two Ca(2+) sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb(+) was not detected at the expected Na(+) site. In kinetic experiments, Na(+) increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca(2+), Na(+) had a negligible effect. Ca(2+) increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na(+) and by ∼82-fold in the presence of Na(+). In molecular-dynamics simulations, Na(+) stabilized the two Na(+)-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca(2+) stabilized the Ca(2+)-binding loop (the 70-loop) and Na(+)-binding loops but not the TF-binding region. Na(+) and Ca(2+) together stabilized both the Na(+)-binding and Ca(2+)-binding loops and the TF-binding region. Previously, Rb(+) has been used to define the Na(+) site in thrombin; however, it was unsuccessful in detecting the Na(+) site in FVIIa. A conceivable explanation for this observation is provided.