Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, 600 025, India.
AU-KBC Research Centre, Anna University, Chennai, Tamil Nadu, 600 044, India.
J Mol Model. 2021 Feb 1;27(2):63. doi: 10.1007/s00894-020-04643-7.
Structural flexibility of the peptide linker connecting two domains is essential for the functioning of multi-domain complex. Nitric oxide synthase (NOS) isoforms contain the oxygenase and the reductase domains connected by calmodulin binding linker (CBL) region. Additionally, the endothelial NOS (eNOS) isoform contain an auto-inhibitory loop (AI) in the FMN reductase sub-domain which represses the inter-domain electron transfer process. Binding of Ca-Calmodulin complex on the CBL region relieves the AI loop repression and facilitates electron transfer from FMN in the reductase domain to the heme in the oxygenase domain. Few experimental studies have reported that in vitro mutation of Serine-615 (S615D) and Serine-633 (S633D) in the FMN reductase sub-domain to aspartic acid increased NO production and increased Ca sensitivity. To understand the role of AI loop in eNOS repression and activation in serine mutants (S615D and S633D), we modelled the FMN reductase sub-domain of human eNOS protein with and without the CBL region. Molecular dynamics simulations performed indicated that the mutant protein AI loop structure was stabilized by salt bridge formed between D615 and R602. It was also found that mutation increased the flexibility of C-terminal residues of eNOS CBL region. The hinge-like movement of the AI loop allowed rotation of the FMN sub-domain clockwise which may favour electron-transfer in the mutant protein. This study provides insight on mutation (S615D and S633D) induced changes in AI loop and increased flexibility of CBL region which may lead to the protein activation and may also facilitate Calmodulin binding at physiological Ca concentration. Graphical Abstract Mutation of amino acid residues contribute to structural changes at molecular level leading to alteration in protein dynamics and its function. Serine-615 and Serine-633 in the auto-inhibitory loop of human eNOS reductase model was mutated to aspartic acid in silico and molecular dynamics simulations of the protein showed that steric hindrance due to mutation altered the auto-inhibitory loop rearrangement and the FMN sub-domain movement favouring electron transfer.
连接两个结构域的肽连接的结构灵活性对于多结构域复合物的功能至关重要。一氧化氮合酶（NOS）同工型包含氧合酶和还原酶结构域，通过钙调蛋白结合连接区（CBL）连接。此外，内皮型一氧化氮合酶（eNOS）同工型在 FMN 还原酶亚结构域中包含一个自动抑制环（AI），抑制了结构域间的电子转移过程。Ca-Calmodulin 复合物与 CBL 区域的结合解除了 AI 环的抑制，促进了电子从还原酶结构域中的 FMN 转移到氧合酶结构域中的血红素。几项实验研究表明，体外突变 FMN 还原酶亚结构域中的丝氨酸 615（S615D）和丝氨酸 633（S633D）为天冬氨酸可增加 NO 产量并增加 Ca 敏感性。为了了解 AI 环在 eNOS 抑制和丝氨酸突变体（S615D 和 S633D）中的激活作用，我们构建了带有和不带有 CBL 区域的人 eNOS 蛋白 FMN 还原酶亚结构域模型。分子动力学模拟表明，突变蛋白的 AI 环结构通过 D615 和 R602 之间形成的盐桥稳定。还发现突变增加了 eNOS CBL 区域 C 端残基的灵活性。AI 环的铰链样运动使 FMN 亚结构域顺时针旋转，这可能有利于突变蛋白中的电子转移。这项研究提供了关于突变（S615D 和 S633D）引起的 AI 环变化和 CBL 区域增加的灵活性的见解，这可能导致蛋白质激活，并且还可能促进生理 Ca 浓度下钙调蛋白的结合。
图形摘要
氨基酸残基的突变导致分子水平上的结构变化，从而改变蛋白质动力学及其功能。在人 eNOS 还原酶模型的自动抑制环中的丝氨酸 615 和丝氨酸 633 被计算机模拟突变为天冬氨酸，该蛋白的分子动力学模拟表明，由于突变引起的空间位阻改变了自动抑制环的重排和 FMN 亚结构域的运动，有利于电子转移。
