Xia Yuan-Ling, Sun Jian-Hong, Ai Shi-Meng, Li Yi, Du Xing, Sang Peng, Yang Li-Quan, Fu Yun-Xin, Liu Shu-Qun
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University Kunming Yunnan P. R. China
Department of Applied Mathematics, Yunnan Agricultural University Kunming Yunnan P. R. China.
RSC Adv. 2018 Aug 22;8(52):29698-29713. doi: 10.1039/c8ra05845h. eCollection 2018 Aug 20.
To investigate the role of electrostatics in different temperature adaptations, we performed a comparative study on subtilisin-like serine proteases from psychrophilic sp. PA-44 (VPR), mesophilic () (PRK), and thermophilic (AQN) using multiple-replica molecular dynamics (MD) simulations combined with continuum electrostatics calculations. The results reveal that although salt bridges are not a crucial factor in determining the overall thermostability of these three proteases, they on average provide the greatest, moderate, and least electrostatic stabilization to AQN, PRK, and VPR, respectively, at the respective organism growth temperatures. Most salt bridges in AQN are effectively stabilizing and thus contribute to maintaining the overall structural stability at 343 K, while nearly half of the salt bridges in VPR interconvert between being stabilizing and being destabilizing, likely aiding in enhancing the local conformational flexibility at 283 K. The individual salt bridges, salt-bridge networks, and calcium ions contribute differentially to local stability and flexibility of these three enzyme structures, depending on their spatial distributions and electrostatic strengths. The shared negatively charged surface potential at the active center of the three enzymes may provide the active-center flexibility necessary for nucleophilic attack and proton transfer. The differences in distributions of the electro-negative, electro-positive, and electro-neutral potentials, particularly over the back surfaces of the three proteases, may modulate/affect not only protein solubility and thermostability but also structural stability and flexibility/rigidity. These results demonstrate that electrostatics contributes to both heat and cold adaptation of subtilisin-like serine proteases through fine-tuning, either globally or locally, the structural stability and conformational flexibility/rigidity, thus providing a foundation for further engineering and mutagenesis studies.
为了研究静电在不同温度适应性中的作用，我们使用多重复制分子动力学（MD）模拟结合连续介质静电计算，对来自嗜冷菌sp. PA - 44（VPR）、嗜温菌（）（PRK）和嗜热菌（AQN）的枯草杆菌蛋白酶样丝氨酸蛋白酶进行了比较研究。结果表明，虽然盐桥并非决定这三种蛋白酶整体热稳定性的关键因素，但在各自的生物体生长温度下，它们平均分别为AQN、PRK和VPR提供了最大、中等和最小的静电稳定性。AQN中的大多数盐桥具有有效的稳定作用，因此有助于在343 K下维持整体结构稳定性，而VPR中近一半的盐桥在稳定和不稳定之间相互转换，这可能有助于增强283 K下的局部构象灵活性。单个盐桥、盐桥网络和钙离子对这三种酶结构的局部稳定性和灵活性的贡献各不相同，这取决于它们的空间分布和静电强度。三种酶活性中心共享的带负电荷表面电位可能为亲核攻击和质子转移提供所需的活性中心灵活性。电负、电正和电中性电位分布的差异，特别是在这三种蛋白酶的背面，可能不仅调节/影响蛋白质的溶解度和热稳定性，还影响结构稳定性和灵活性/刚性。这些结果表明，静电通过全局或局部微调结构稳定性和构象灵活性/刚性，有助于枯草杆菌蛋白酶样丝氨酸蛋白酶对热和冷的适应，从而为进一步的工程和诱变研究奠定了基础。
