Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

设计型锚蛋白重复蛋白（Designed Ankyrin Repeat Proteins，简称DARPin）是一类新型结合蛋白，可通过筛选与进化获得对靶标高亲和力、高特异性的结合能力。本研究聚焦于DARPin H10-2-G3，该蛋白经进化后可与人类表皮生长因子受体2（HER2）以极高亲和力结合。研究表明，30%的乳腺癌患者存在HER2过表达情况，HER2亦是FDA批准的治疗性单克隆抗体曲妥珠单抗（trastuzumab）、帕妥珠单抗（pertuzumab）以及小分子酪氨酸激酶抑制剂的作用靶标。本研究采用计算大分子对接技术，并结合形状互补性、相互作用能、静电互补性等多种界面评价指标，构建DARPin H10-2-G3与HER2复合物的三维结构模型。本研究对二者的结合界面进行了分析，并通过实验验证了该结构模型：在H10-2-G3与HER2的推定结合界面处引入的HER2点突变可使结合亲和力最高下降100倍，且不会影响曲妥珠单抗的结合能力。后续将该模型与已解析的复合物X射线晶体结构进行比对，发现其主链原子均方根偏差为0.84~1.14埃（Å）。本研究证实了结构生物信息学计算技术在构建蛋白质-蛋白质相互作用有效结构模型方面的应用能力。