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

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 77; no. 6; pp. 809 - 819
• Main Authors: Vadivel, Kanagasabai, Schmidt, Amy E, Cascio, Duilio, Padmanabhan, Kaillathe, Krishnaswamy, Sriram, Brandstetter, Hans, Bajaj, S Paul
• Format: Journal Article
• Language: English
• Published: Chester Wiley Subscription Services, Inc 01.06.2021
• Subjects: Binding; Calcium; Calcium ions; Coagulation; Coagulation factor VIIa; Domains; Epidermal growth factor; Growth factors; Ions; Magnesium; Protease; Proteinase; Rubidium; Sodium; Substrates; Thrombin; Tissue factor
• ISSN: 0907-4449; 1399-0047
• DOI: 10.1107/S2059798321003922

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.