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^[2]) and the third was found on the surface. . The FVIIa light chain is in blue and the heavy chain is in red. The sTF is shown in magenta. The active site residue Ser195 is shown in space filling, and benzamidine (Bz) bound at the active site is shown in stick representation. The Ca²⁺, Mg²⁺ and Rb⁺ bound to FVIIa are shown as green, orange and purple spheres, respectively. Note that Ca²⁺ at position 3 and at position 5 are replaced by Rb⁺ 1 and Rb⁺ 2, respectively. 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^[3]). While the absence of a sodium ion in 1klj is consistent with its limited 2.44 Å resolution, the data set 1kli, determined at 1.7 Å 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^[4]), 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. in the absence of TF (PDBID 1kli). The FVIIa residues Tyr184 {332}, Ser185 {333}, Thr221 {370} and His224 {373} carbonyl oxygen atoms and the two water molecules that serve as ligands for Na+ are shown. The sodium (Na) and water molecules are shown as purple and red spheres, respectively.

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 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^[5]) 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, . In FVIIa, residues from both the 184 and 220 loops (Y184, S185, T221 and H224) participate in coordinating to Na⁺, whereas in thrombin only residues from the 220-loop (R221A and K224) are involved. The Na⁺ and Rb⁺ are shown as pink and purple spheres, respectively. The residues that serve as the ligands for Na+ are shown in stick representation. The residue 225, which defines the presence of a Na⁺ site in these proteases is also shown in stick representation. The FVIIa loops are shown in green and the thrombin loops in yellow. The four residue insert in the 184-loop of thrombin is shown in magenta. 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^[6][7]). 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.

PDB reference: factor VIIa–soluble tissue factor complex, 4ibl.

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
2. ↑ Soriano-Garcia M, Padmanabhan K, de Vos AM, Tulinsky A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry. 1992 Mar 10;31(9):2554-66. PMID:1547238
3. ↑ Sichler K, Banner DW, D'Arcy A, Hopfner KP, Huber R, Bode W, Kresse GB, Kopetzki E, Brandstetter H. Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions. J Mol Biol. 2002 Sep 20;322(3):591-603. PMID:12225752
4. ↑ Joosten RP, Long F, Murshudov GN, Perrakis A. The PDB_REDO server for macromolecular structure model optimization. IUCrJ. 2014 May 30;1(Pt 4):213-20. doi: 10.1107/S2052252514009324. eCollection, 2014 Jul 1. PMID:25075342 doi:http://dx.doi.org/10.1107/S2052252514009324
5. ↑ Shannon, R. D. (1976). Acta Cryst. A32, 751-767
6. ↑ Machius M, Declerck N, Huber R, Wiegand G. Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad. Structure. 1998 Mar 15;6(3):281-92. PMID:9551551
7. ↑ Nonaka T, Fujihashi M, Kita A, Hagihara H, Ozaki K, Ito S, Miki K. Crystal structure of calcium-free alpha-amylase from Bacillus sp. strain KSM-K38 (AmyK38) and its sodium ion binding sites. J Biol Chem. 2003 Jul 4;278(27):24818-24. Epub 2003 Apr 28. PMID:12719434 doi:10.1074/jbc.M212763200