A DE NOVO PROTEIN BINDING PAIR BY COMPUTATIONAL DESIGN AND DIRECTED EVOLUTION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白质-蛋白质界面的从头设计是对我们对蛋白质-蛋白质相互作用基本原理的理解的严格测试，并将使独特的方法能够应对生物和医学挑战。在这里，我们描述了一种基于Motif的方法来计算设计具有天然界面组成和相互作用密度的蛋白质-蛋白质复合体。使用这种方法，我们设计了一对蛋白质，pRB和PDAR，它们的Kd值为130 nm，比以前设计的从头蛋白质-蛋白质复合体紧密1000倍。定向进化发现了两个点突变，它们提高了对180 PM的亲和力亲和力成熟的配合物的晶体结构表明，结合完全是通过设计的界面残基进行的。令人惊讶的是，在体外进化的复合体中，其中一个伙伴相对于原始设计模型旋转了180度，但仍然保持了中央计算设计的热点相互作用，并保留了许多外围相互作用的特征。这项工作表明，通过在两个非相互作用的伙伴上设计互补的相互作用面，可以创建高亲和力的蛋白质界面，并强调了剩余的挑战。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180 degrees relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.