Computational Design of Protein-Protein Interactions

描述（由申请人提供）：蛋白质-蛋白质相互作用对几乎所有生物过程都是必不可少的。具有新的结合特性的工程蛋白是细胞和分子研究的重要工具，在有利的情况下可以用作治疗剂。本研究的目的是开发和测试设计新的蛋白质-蛋白质相互作用的计算方法。这是一个具有挑战性的问题，原因有很多：给定两种蛋白质，我们可能不清楚它们应该如何对接以促进结合，许多蛋白质在结合时经历侧链和主链重排，必须通过设计跨界面的有利相互作用来克服大的脱溶自由能。为了解决这些问题，我们将测试三种设计策略，利用罗塞塔分子建模程序，并基于在自然发生的蛋白质相互作用中观察到的结构特征。在目标1中，我们将设计由相互作用-链介导的复合物。我们将选择具有溶剂暴露链的支架蛋白，并将其用于同型二聚体或异源二聚体设计。两种蛋白质边缘链之间的氢键将建立蛋白质的相对定位，并补偿解离能。相互作用链周围残基的序列优化和主链优化将用于进一步稳定相互作用。在目标2中，我们将使用金属结合来模板蛋白质-蛋白质相互作用。形成锌结合位点一半的氨基酸对将构建在一个（设计同型二聚体）或两个蛋白质（设计异源二聚体）的表面上。然后蛋白质将彼此对接形成金属结合位点，周围的残基将被重新设计以在界面上形成有利的相互作用。金属结合将提供亲和力和特异性的目标相互作用。在目标3中，我们将研究环介导的相互作用。纤维连接蛋白结构域的表面环将被重新设计以结合靶蛋白。循环构象和序列的迭代优化将用于寻找与伙伴形成有利相互作用的低能序列/结构对。为了降低这个问题的复杂性，我们将首先考虑一些情况，其中一个纤维连接蛋白环不是从头开始设计的，而是基于已知的与目标蛋白结合的序列。为了实现这三个目标，我们将使用生物物理结合测量、定点诱变和高分辨率结构测定（核磁共振或x射线）来评估计算预测。通过这个项目，我们将扩展计算蛋白质设计的能力，并测试我们对蛋白质-蛋白质界面亲和力和特异性的主要决定因素的理解。

DESCRIPTION (provided by applicant): Protein-protein interactions are essential to almost all biological processes. Engineered proteins with novel binding properties are important tools for cellular and molecular research and can be used as therapeutic agents in favorable cases. The objective of this research is to develop and test computational methods for designing new protein-protein interactions. This is a challenging problem for many reasons: given two proteins it may not be clear how they should be docked to promote binding, many proteins undergo side chain and backbone rearrangement upon binding, and large free energies of desolvation must be overcome by designing favorable interactions across the interface. To address these issues, we will test three design strategies that make use of the Rosetta molecular modeling program and are based on structural features observed in naturally occurring protein interactions. In Aim 1, we will design complexes that are mediated by interacting -strands. We will choose scaffold proteins with solvent-exposed -strands and use them for either homodimer or heterodimer design. Hydrogen bonding between the edge strands of the two proteins will establish the relative positioning of the proteins and compensate for desolvation energies. Sequence optimization and backbone refinement of residues surrounding the interacting strands will be used to further stabilize the interaction. In Aim 2, we will use metal binding to template protein-protein interactions. Pairs of amino acids that form one-half of a zinc-binding site will be built onto the surface of one (to design homodimers) or two proteins (to design heterodimers). The proteins will then be docked against each other to form the metal binding site and the surrounding residues will be redesigned to form favorable interactions across the interface. Metal binding will provide both affinity and specificity to the target interaction. In Aim 3, we will examine loop- mediated interactions. Surface loops on the fibronectin domain will be redesigned to bind target proteins. Iterative optimization of loop conformation and sequence will be used to search for low energy sequence/structure pairs that form favorable interactions with the partner. To lower the complexity of this problem, we will first consider cases where one of the fibronectin loops is not designed from scratch, but rather is based on sequences that are already known to bind the target protein. For all three aims we will use biophysical binding measurements, site-directed mutagenesis and high-resolution structure determination (NMR or X-ray) to evaluate the computational predictions. By pursuing this project we will extend the capabilities of computational protein design and test our understanding of the primary determinants of affinity and specificity at protein-protein interfaces.