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 Mg²⁺ ions and four Ca²⁺ ions in the GLA domain, one Ca²⁺ ion in the EGF1 domain and one Ca²⁺ 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 Mg²⁺, Ca²⁺ and Rb⁺ ions. In this structure, Rb⁺ replaced two Ca²⁺ 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 Ca²⁺, Na⁺ had a negligible effect. Ca²⁺ 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. Ca²⁺ stabilized the Ca²⁺-binding loop (the 70-loop) and Na⁺-binding loops but not the TF-binding region. Na⁺ and Ca²⁺ together stabilized both the Na⁺-binding and Ca²⁺-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.