Journal:Acta Cryst D:S2059798321003922

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

K. Vadivel, A. E. Schmidt, D. Cascio, K. Padmanabhan, S. Krishnaswamy, H. Brandstetter and S. P. Bajaj ^[1]

Molecular Tour
Identification of the metal binding sites in proteins is important to understand their functions in biology. The role of divalent metal ions Ca2+ and Mg2+ are well established for coagulation factor (F) VIIa but not the role of monovalent ion Na+. Coagulation FVIIa consists of a gamma-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. Here, we performed structural, kinetic, and molecular dynamics studies to investigate the role of Na+ in FVIIa structure and function. The FVIIa/soluble tissue factor (sTF) complex was crystallized in the presence of Ca2+, Mg2+ and Rb+ and the data were collected near the Rb K absorption edge to examine whether Rb+ can occupy the Na+-site in FVIIa. The FVIIa/sTF structure was determined by molecular replacement and the structure is similar to the previous FVIIa/sTF complex structure. Based upon the Rb anomalous signal, three Rb+ were found in the GLA domain and three in the protease domain. Two of the three Rb+ in the GLA domain occupied the Ca2+-binding sites at positions 3 and 5 (metal binding sites numbering in the GLA domain based on Tulinsky and coworkers, Sorano-Garcia et al., 1992) and the third was found on the surface (Fig. 1). Moreover, although three Rb+ were identified in the protease domain, but none at the putative Na+-site and all were surface bound. In kinetic experiments, Na+ increased the FVIIa amidolytic activity towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ~20-fold; however, in the presence of Ca2+, sodium had a negligible effect. In molecular dynamics simulations, sodium stabilized the two Na+-binding loops (184-loop and 220-loop) and the TF-binding region spanning residues 163-180 (chymotrypsin numbering) in FVIIa. Thus Na+, in part, contributes towards stabilization of the FVIIa protease domain. In this context, it is particularly interesting to reinvestigate crystal structures of FVIIa which were determined in the absence of TF, particularly pdb entries 1klj and 1kli, each missing a Na+ at the expected sodium-binding site (Sichler et al., 2002). While the absence of a sodium ion in 1klj is consistent with its limited 2.44 Å resolution, the data set 1kli, determined at 1.7Å resolution, deserves a more careful analysis. Indeed, the relevant solvent structure is intriguing. According to the 1kli coordinate set, a water molecule is positioned in the neighborhood to the three carbonyl oxygens of Tyr184, Thr221 and His224. Such a three carbonyl oxygen coordination is inconsistent with an ordered water molecule, but consistent with a sodium ion. Furthermore, current structure refinement protocols, including the automatic PDB_REDO (Joosten et al., 2014), revealed a significant positive difference electron density at more than 5 σ above the mean. Consequently, a re-analysis with current refinement protocols strongly favors the presence of a sodium ion in FVIIa in the absence of TF (Fig. 2). Previously, Rb+ was used as a probe to identity the Na+ site in thrombin but it was unsuccessful in case of FVIIa. A possible explanation for the absence of Rb+ occupancy at the Na+ site in FVIIa is the nature of Na+ site, which differs from thrombin. In FVIIa, both 184- and 220-loops provide coordination ligands for Na+, whereas in thrombin, only the 220-loop is involved. Notably, the Na+ site in thrombin is located at the prominent water channel filled with more than 20 conserved water molecules that is deep and exposed to the surface. As a result, it allows Rb+ to occupy the Na+ site even though Rb+ has larger ionic radius (1.52 Å, Shannon, 1976) and requires longer coordination distance as compared to the Na+ (ionic radius 1.02 Å). In contrast, the Na+ site in FVIIa is narrow and less exposed to the surface. Thus, spatial restrains imposed by the 184 and 220-loops in FVIIa prevent Na+ site to be occupied by Rb+ due to its larger ionic radius compared to the Na+ (Fig. 3). This observation is consistent with an earlier finding that Rb+ does not always occupy the Na+ site in macromolecules, especially at less exposed and narrow spaces (Machius et al., 1998; Nonaka et al., 2003). Thus, molecular environment of the Na+ site in a protein determines whether or not Rb+ can occupy the Na+ site. Overall, the analysis points out that the Na+ site in FVIIa is similar to that in FIXa, FXa and APC but not to thrombin. The Na+ site, in conjunction with Ca2+ primarily plays a structural role by stabilizing the FVIIa protease domain.

References:

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References

1. ↑ Vadivel K, Schmidt AE, Cascio D, Padmanabhan K, Krishnaswamy S, Brandstetter H, Bajaj SP. Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium. Acta Crystallogr D Struct Biol. 2021 Jun 1;77(Pt 6):809-819. doi:, 10.1107/S2059798321003922. Epub 2021 May 14. PMID:34076594 doi:http://dx.doi.org/10.1107/S2059798321003922