Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
J Chem Theory Comput. 2022 Mar 8;18(3):1982-2001. doi: 10.1021/acs.jctc.1c01187. Epub 2022 Feb 16.
Protein-protein association is often mediated by electrostatic interactions and modulated by pH. However, experimental and computational studies have often overlooked the effect of association on the protonation state of the protein. In this work, we present a methodological approach based on constant-pH molecular dynamics (MD), which aims to provide a detailed description of a pH-dependent protein-protein association, and apply it to the dimerization of β-lactoglobulin (BLG). A selection of analyses is performed using the data generated by constant-pH MD simulations of monomeric and dimeric forms of bovine BLG, in the pH range 3-8. First, we estimate free energies of dimerization using a computationally inexpensive approach based on the Wyman-Tanford linkage theory, calculated in a new way through the use of thermodynamically based splines. The individual free energy contribution of each titratable site is also calculated, allowing for identification of relevant residues. Second, the correlations between the proton occupancies of pairs of sites are calculated (using the Pearson coefficient), and extensive networks of correlated sites are observed at acidic pH values, sometimes involving distant pairs. In general, strongly correlated sites are also slow proton exchangers and contribute significantly to the pH-dependency of the dimerization free energy. Third, we use ionic density as a fingerprint of protein charge distribution and observe electrostatic complementarity between the monomer faces that form the dimer interface, more markedly at the isoionic point (where maximum dimerization occurs) than at other pH values, which might contribute to guide the association. Finally, the pH-dependent dimerization modes are inspected using PCA, among other analyses, and two states are identified: a at pH 4-8 (with the typical alignment of the crystallographic structure) and a at pH 3-4 (with a tighter association and rotated alignment). This work shows that an approach based on constant-pH MD simulations can produce rich detailed pictures of pH-dependent protein associations, as illustrated for BLG dimerization.
蛋白质-蛋白质的相互作用通常是通过静电相互作用介导的，并受 pH 值调节。然而，实验和计算研究往往忽略了相互作用对蛋白质质子化状态的影响。在这项工作中，我们提出了一种基于恒 pH 分子动力学 (MD) 的方法，旨在提供对 pH 依赖性蛋白质-蛋白质相互作用的详细描述，并将其应用于β-乳球蛋白 (BLG) 的二聚化。通过对牛 BLG 单体和二聚体形式在 pH 值为 3-8 的范围内进行恒 pH MD 模拟产生的数据，进行了一系列分析。首先，我们使用基于 Wyman-Tanford 键合理论的计算成本较低的方法来估计二聚化的自由能，该方法通过使用热力学基础样条曲线以新的方式计算。还计算了每个可滴定位点的个体自由能贡献，从而可以识别相关残基。其次，计算了对位点对的质子占有率之间的相关性（使用 Pearson 系数），并且在酸性 pH 值下观察到广泛的相关位点网络，有时涉及到遥远的对。通常，强相关的位点也是快速质子交换体，并且对二聚化自由能的 pH 依赖性有重要贡献。第三，我们使用离子密度作为蛋白质电荷分布的指纹，并观察到形成二聚体界面的单体表面之间的静电互补性，在等电点（最大二聚化发生的位置）比在其他 pH 值下更为明显，这可能有助于指导相互作用。最后，通过主成分分析 (PCA) 等分析方法检查 pH 依赖性的二聚化模式，并确定了两种状态：一种是在 pH 4-8（具有典型的晶体结构排列），另一种是在 pH 3-4（具有更紧密的结合和旋转排列）。这项工作表明，基于恒 pH MD 模拟的方法可以产生丰富的 pH 依赖性蛋白质相互作用的详细图片，如图 1 所示，BLG 二聚化就是一个很好的例子。
