Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium,Acta Crystallographica Section D

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Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium
Acta Crystallographica Section D ( IF 2.6 ) Pub Date : 2021-05-24 , DOI: 10.1107/s2059798321003922
Kanagasabai Vadivel ₁ , Amy E Schmidt ₂ , Duilio Cascio ₃ , Kaillathe Padmanabhan ₄ , Sriram Krishnaswamy ₅ , Hans Brandstetter ₆ , S Paul Bajaj ₁

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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.

中文翻译：

含钙、镁和铷的人因子 VIIa-可溶性组织因子的结构

凝血因子 VIIa (FVIIa) 由一个 γ-羧基谷氨酸 (GLA) 结构域、两个表皮生长因子样 (EGF) 结构域和一个蛋白酶结构域组成。FVIIa在GLA结构域中结合3个Mg ²⁺离子和4个Ca ²⁺离子，在EGF1结构域中结合1个Ca ²⁺离子，在蛋白酶结构域中结合1个Ca ^2+离子。此外，FVIIa在蛋白酶结构域中含有Na ^+位点。由于Na ⁺和水共享相同数量的电子，因此蛋白质中的Na ⁺位点很难与X 射线结构中的水区分开。在这里，为了验证 FVIIa 中的 Na ^{+位点，以 1.8 Å 分辨率解析了包含 Mg}²⁺、Ca ²⁺和 Rb ⁺离子的 FVIIa-可溶性组织因子 (TF) 复合物的结构。在该结构中，Rb ⁺取代了GLA结构域中的两个Ca ²⁺位点，并占据了蛋白酶结构域中的三个非金属位点。^{然而，在预期的Na +}位点没有检测到Rb ⁺。在动力学实验中，Na ^{+使 FVIIa 对合成底物 S-2288 (H-}d -Ile-Pro-Arg -p-硝基苯胺)的酰胺分解活性增加约 20 倍；^{然而，在Ca 2+}存在的情况下，Na ^{+ 的}影响可以忽略不计。Ca ^{2+使 FVIIa 对 S-2288 的水解活性在不存在 Na}⁺的情况下增加约 60 倍，在存在 Na ⁺的情况下增加约 82 倍。在分子动力学模拟中，Na ⁺稳定了两个 Na ⁺结合环（184 环和 220 环）和跨越残基 163-180 的 TF 结合区域。Ca ²⁺稳定Ca ²⁺结合环（70 环）和Na ⁺结合环，但不稳定TF 结合区域。Na ⁺和Ca ²⁺一起稳定Na ⁺结合环和Ca ²⁺结合环以及TF 结合区域。此前，Rb ⁺已被用来定义凝血酶中的Na ^+位点；然而，未能成功检测到FVIIa中的Na ⁺位点。对此观察结果提供了一个可以想象的解释。

更新日期：2021-06-02

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