Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 3¹⁰ helix between helix H and strand 2 of β-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

中文翻译：

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肝素通过静电排斥阻断 Kallistatin 对组织激肽释放酶 1 的抑制。

Kallistatin，也称为 SERPINA4，由于其对组织激肽释放酶 1 (KLK1) 的特异性抑制和/或其肝素结合能力，与血压和血管生成的调节有关。肝素与卡利他汀的结合已被证明可以阻断卡利他汀对 KLK1 的抑制，但这种阻断背后的详细分子机制尚不清楚。在这里，我们分别以 1.9 和 1.8 Å 的分辨率解析了人卡利他汀及其与肝素复合物的晶体结构。结构显示卡利他汀具有保守的丝氨酸蛋白酶抑制剂折叠，并在反应性环裂解时经历典型的应激-松弛构象变化。结构分析和诱变研究表明，kallistatin 的肝素结合位点位于具有正静电势的表面上，靠近 H 螺旋和 β-sheet C 2 链之间独特的突出 3 10^螺旋。肝素在此位点上的结合将阻止 KLK1由于肝素与 KLK1 带负电的表面之间的静电排斥作用，与 kallistatin 对接，从而阻断 kallistatin 对 KLK1 的抑制作用。将 KLK1 的酸性外位点 1 残基替换为凝血酶中的碱性氨基酸会导致加速抑制。总而言之，这些数据表明肝素控制了卡利他汀的特异性，使得微循环内 KLK1 产生的激肽将通过卡利他汀与内皮细胞的肝素样糖胺聚糖的结合而受到局部保护。