Journal:Acta Cryst D:S2059798321003922
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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<ref name="Sichler">PMID:12225752</ref>). 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<ref name="Joosten">PMID:25075342</ref>), 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. <scene name='88/883793/Cv/8'>Na+ site in FVIIa protease domain</scene> 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. | 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<ref name="Sichler">PMID:12225752</ref>). 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<ref name="Joosten">PMID:25075342</ref>), 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. <scene name='88/883793/Cv/8'>Na+ site in FVIIa protease domain</scene> 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 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<ref name="Shannon">Shannon, R. D. (1976). Acta Cryst. A32, 751-767</ref>) 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, <scene name='88/883793/Cv/14'> | + | 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<ref name="Shannon">Shannon, R. D. (1976). Acta Cryst. A32, 751-767</ref>) 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, <scene name='88/883793/Cv/14'>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+</scene>. 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 spatial restrains imposed by the 184 and 220 loops prevent Rb+ occupancy at the Na+ site in FVIIa. The Na+ and Rb+ are shown as pink and purple spheres, respectively. The ionic radii of Na+ (1.02 Å) and Rb+ (1.52 Å) are used to draw the spheres (Shannon, 1976<ref name="Shannon">Shannon, R. D. (1976). Acta Cryst. A32, 751-767</ref>). 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<ref name="Machius">PMID:9551551</ref><ref name="Nonaka">PMID:12719434</ref>). 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. |
<b>References</b><br> | <b>References</b><br> | ||
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