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Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium
Author(s) -
Vadivel Kanagasabai,
Schmidt Amy E.,
Cascio Duilio,
Padmanabhan Kaillathe,
Krishnaswamy Sriram,
Brandstetter Hans,
Bajaj S. Paul
Publication year - 2021
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s2059798321003922
Subject(s) - chemistry , tissue factor , protease , calcium , factor x , magnesium , binding site , biophysics , stereochemistry , enzyme , crystallography , coagulation , biochemistry , thrombin , platelet , biology , psychology , organic chemistry , psychiatry , immunology
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.