Computational Alanine Scanning MutagenesisAn Improved Methodological Approach for Protein–DNA Complexes

Proteins and protein-based complexes are the basis of many key systems in nature and have been the subject of intense research in the last decades, in an attempt to acquire comprehensive knowledge of reactions that take place in nature. Computational Alanine Scanning Mutagenesis approaches have been extensively used in the study of protein interfaces and in the determination of the most important residues for complex formation, the Hot-spots. However, as it is usually applied to the study of protein–protein interfaces, we tried to modify and apply it to the study of protein–DNA interfaces, which are also crucial in nature but have not been the subject of as much research. In this work, we carry out MD simulations of seven protein–DNA complexes and tested the influence of the variation of different parameters on the determination of the binding free energy terms (ΔΔGbinding) of 78 mutations: solvent representation, internal dielectric constant, Linear and Nonlinear Poisson–Boltzmann equation, Generalized Born model, simulation time, number of structures analyzed, number of MD trajectories, force field used, and energetic terms involved. Overall, this new approach gave an average error of 1.55 kcal/mol, and P, R, F1, accuracy, and specificity values of 0.78, 0.50, 0.61, 0.77, and 0.92, respectively. This improved computational alanine scanning mutagenesis approach may serve as a tool to explore the behavior of this important class of complexes.蛋白质及基于蛋白质的复合物是自然界诸多关键系统的核心基础，近数十年来一直是研究热点，学界旨在全面阐明自然界中发生的各类生化反应机制。计算丙氨酸扫描诱变（Computational Alanine Scanning Mutagenesis）方法已被广泛应用于蛋白质界面研究，以及鉴定复合物形成过程中的关键残基——热点残基（Hot-spots）。然而，该方法通常仅用于蛋白质-蛋白质界面的研究，因此本研究尝试对其进行改进，并将其应用于蛋白质-DNA界面的研究；此类界面在自然界中同样发挥关键作用，但相关研究仍相对匮乏。本研究对7组蛋白质-DNA复合物开展分子动力学（MD, Molecular Dynamics）模拟，并针对78个突变体的结合自由能变化量（ΔΔGbinding）计算，系统考察了多项参数对结果的影响，包括溶剂表征方式、内部介电常数、线性与非线性泊松-玻尔兹曼（Poisson–Boltzmann）方程、广义玻恩（Generalized Born）模型、模拟时长、分析的结构数量、分子动力学轨迹数量、所用力场以及涉及的能量项。整体而言，该改进方法的平均误差为1.55 kcal/mol，其精确率（P）、召回率（R）、F1值、准确率及特异性分别为0.78、0.50、0.61、0.77与0.92。该改进后的计算丙氨酸扫描诱变方法，可作为探究此类重要复合物行为的有效工具。