De novo design of small-molecule-binding proteins

项目摘要 蛋白质-配体结合事件贯穿所有生命过程。蛋白质设计测试并扩展了我们对 蛋白质的折叠和功能是通过从头开始创造蛋白质来实现的。该建议旨在制定一项 用于设计以高亲和力结合任何小分子的蛋白质的计算方法， 选择性配体结合蛋白质设计的最新技术严重依赖于随机实验 优化和筛选。如果我们真正了解蛋白质如何结合小分子，我们应该能够 直接从计算机模型到紧密的活页夹。推动这一提议的假设是，蛋白质使用一种 在整个进化过程中，大量但现在数量可观的分子相互作用基序组合起来， 现代蛋白质的结合位点。将采用计算方法来揭示这一组 蛋白质数据库（PDB）中的蛋白质结构的大型数据库中的相互作用。结合位点 将通过将配体的所有官能团的基序取样到蛋白质骨架上来设计。我们称之 结合位点的聚合基序（Convergent Motifs for Binding Sites，COMBS）COMBS被用于设计ABLER，第一个 一种从头开始设计的配体结合蛋白，它能与抗血栓药物阿哌沙班结合， 前所未有的高亲和力，无需实验优化序列。ABLER具有潜在的临床意义 作为抗凝血解毒剂的相关性，尽管这超出了提案的范围。高分辨率晶体 ABLER的结构与设计模型一致，无论是在整体拓扑结构还是预期的分子结构 与配体的相互作用。该提案的目的1集中于设计ABLER的变体以增加亲和力 并探测观察到的药物-蛋白质相互作用的分子基础。目标2着重于水在以下方面的作用： 配体结合蛋白质设计，由晶体中发现的水介导的蛋白质-配体相互作用激发 结构在这个目标中，我将从PDB中整理一个水蛋白相互作用的数据库，并使用这些数据库来 在设计过程中对水介导的蛋白质-配体相互作用进行采样。我也要学会用清水 分子动力学模拟，以严格评估水在结合中的作用。Aim 3使用COMBS重新设计 吡咯赖氨酸tRNA合成酶的结合位点，用于掺入带电荷的非天然氨基酸（如 磺基酪氨酸）进入哺乳动物细胞，因为迄今为止， 不成功。这些目标将增加我在分子生物学，计算蛋白质设计， 蛋白质结构表征（X射线晶体学和NMR）。本工作的K99部分 DeGrado实验室将让我接触到科学过程的各个方面，从开始到出版。比尔是一个世界 他是蛋白质设计方面的专家，他的洞察力对该项目的成功至关重要。在加州大学旧金山分校， 我定期与伊森韦斯会面，他带来了一个医生科学家的观点， 抗血栓研究和临床应用历史悠久。我与UCSF教授雷的合作 王将使我接触到非天然氨基酸掺入领域，并将对应用COMBS至关重要 翻译后修饰模拟物的最有效靶点。拟议的研究和培训 这将大大补充我目前的技能和背景，这将是我的研究必不可少的 在我转变为独立首席研究员的过程中，

PROJECT SUMMARY Protein-ligand binding events underlay all life processes. Protein design tests and extends our knowledge of protein folding and function through the creation of proteins from scratch. This proposal aims to develop a computational method for the design of proteins that bind to any small molecule with high affinity and selectivity. The state-of-the-art in ligand-binding protein design critically relies on random experimental optimization and screening. If we truly understand how proteins bind small molecules, we should be able to go directly from computer models to tight binders. The hypothesis that drives this proposal is that proteins use a vast but now enumerable number of molecular interaction motifs combinatorially throughout evolution to create the binding sites of modern-day proteins. Computational methods will be employed to uncover this set of interactions in the large database of protein structures available in the protein databank (PDB). Binding sites will be designed by sampling motifs for all functional groups of a ligand onto a protein backbone. We call this design method Convergent Motifs for Binding Sites (COMBS). COMBS was used to design ABLER, the first ligand-binding protein designed from scratch to bind its target ligand—the antithrombotic drug apixaban—with an unprecedentedly high affinity, without experimental optimization of sequence. ABLER has potential clinical relevance as an anti-clotting antidote, although that is outside the scope of the proposal. High-resolution crystal structures of ABLER agree with the design model, both in overall topology and the intended molecular interactions with the ligand. Aim 1 of this proposal focuses on designing variants of ABLER to increase affinity and probe the molecular bases for the observed drug-protein interactions. Aim 2 focuses on the role of water in ligand-binding protein design, motivated by the water-mediated protein-ligand interaction found in the crystal structure. In this Aim, I will curate a database of water-protein interactions from the PDB and use these to sample water-mediated protein-ligand interactions during design. I will also learn to use explicit-water molecular dynamics simulations to critically assess the roles of water in binding. Aim 3 uses COMBS to redesign the binding site of pyrrolysine tRNA synthetase for incorporation of charged unnatural amino acids (such as sulfotyrosine) into mammalian cells, since laboratory evolution and library screens for this goal have so far been unsuccessful. These aims will augment my training in molecular biology, computational protein design, and protein structural characterization (X-ray crystallography and NMR). The K99 portion of this work in the DeGrado lab will expose me to all aspects of the scientific process, from inception to publication. Bill is a world expert in protein design, and his insight is critical to the success of the project. At UCSF, I will gain much through my regularly scheduled meetings with Ethan Weiss, who brings the perspective of a physician scientist with a long history of antithrombotic research and clinical applications. My collaboration with UCSF professor Lei Wang will expose me to the field of unnatural amino acid incorporation and will be critical for applying COMBS to the most impactful targets for mimics of post-translational modifications. The research and training proposed herein will greatly complement my current skillset and background, which will be essential to my research program as I transition into an independent principal investigator.