Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

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 ions and four Ca ions in the GLA domain, one Ca ion in the EGF1 domain and one Ca 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, Ca and Rb ions. In this structure, Rb replaced two Ca 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, Na had a negligible effect. Ca 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 stabilized the Ca-binding loop (the 70-loop) and Na-binding loops but not the TF-binding region. Na and Ca together stabilized both the Na-binding and Ca-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.