The National Institute of Advanced Industrial Science and Technology , 1-1-1, Higashi , Tsukuba 305-8566 , Japan.
Daicel Corporation , Innovation Park, 1239, Shinzaike, Aboshi , Himeji , Hyogo 671-1283 , Japan.
ACS Chem Biol. 2019 Dec 20;14(12):2729-2736. doi: 10.1021/acschembio.9b00652. Epub 2019 Oct 22.
Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.
受环境触发因素控制的蛋白质-蛋白质相互作用在各种生物和工业应用中具有巨大的潜力。在本研究中，我们旨在通过结构导向的组氨酸取代方法设计一种依赖 pH 的蛋白质-蛋白质相互作用，该作用利用分子内静电斥力。我们将该策略应用于蛋白 G，即免疫球蛋白 G 的亲和配体，并表明即使单个点突变也能有效地提高结合相互作用的 pH 敏感性，而不会对其结构稳定性或固有结合功能产生不利影响。根据环境的 pH 值，取代的组氨酸与其相邻的带正电荷的残基之间的静电斥力会破坏蛋白质-蛋白质相互作用。在结构上，取代的组氨酸残基位于赖氨酸残基附近，该残基可以与免疫球蛋白 G 形成氢键。从热力学角度来看，引入的静电斥力反映在酸性条件下结合的放热热损失显著增加，而伴随的焓熵补偿部分抑制了 pH 敏感性的提高。因此，工程化的 pH 敏感蛋白 G 可以在酸性条件下实现抗体的纯化。这种分子内设计可以与传统的蛋白质-蛋白质界面设计相结合。此外，这里提出的方法为优化依赖 pH 的分子相互作用提供了额外的设计标准。
