De novo design of small-molecule-binding proteins

小分子结合蛋白的从头设计

• 批准号: 10055537
• 负责人: Nicholas Polizzi
• 金额: $ 9.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055537
• 关键词: Achievement; Affinity; Agreement; Algorithm Design; Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Anticoagulants; Antidotes; Antithrombin III; Award; Binding; Binding Proteins; Binding Sites; Charge; Clinical; Coagulation Process; Collaborations; Complement; Complex; Computer Models; Computing Methodologies; Crystallization; Databases; Development; Drug Delivery Systems; Enzymes; Event; Evolution; Geometry; Goals; Health; Heterogeneity; Human; Hydrogen Bonding; Hydrophobicity; Individual; Knowledge; Laboratories; Learning; Libraries; Life; Ligands; Ligase; Mammalian Cell; Mediating; Medical; Methods; Modernization; Molecular Biology; Molecular Conformation; Molecular Probes; Motivation; Mutagenesis; Nature; Pharmaceutical Preparations; Pharmacologic Substance; Phosphoserine; Physicians; Play; Porphyrins; Post-Translational Protein Processing; Principal Investigator; Process; Protein Engineering; Protein Structure Databases; Proteins; Publications; Recording of previous events; Research; Research Training; Resolution; Roentgen Rays; Role; Sampling; Schedule; Scientist; Set protein; Specificity; Structure; System; Temperature; Testing; Time; Training; Variant; Vertebral column; Water; Work; X-Ray Crystallography; base; clinical application; clinically relevant; combinatorial; data warehouse; design; functional group; infancy; insight; meetings; model design; molecular dynamics; molecular recognition; mutant; novel; phosphohistidine; porphyrin a; professor; programs; protein folding; protein function; protein protein interaction; pyrrolysine; screening; sensor; small molecule; success; tyrosine O-sulfate; unnatural amino acids; water sampling

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.

项目概要 蛋白质-配体结合事件是所有生命过程的基础。蛋白质设计测试并扩展我们的知识 通过从头开始创建蛋白质来进行蛋白质折叠和功能。该提案旨在开发一个 用于设计以高亲和力与任何小分子结合的蛋白质的计算方法 选择性。最先进的配体结合蛋白设计很大程度上依赖于随机实验 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粘合剂。推动这一提议的假设是蛋白质使用 在整个进化过程中，大量但现在可数的分子相互作用基序组合起来创造 现代蛋白质的结合位点。将采用计算方法来揭示这组 蛋白质数据库 (PDB) 中提供的大型蛋白质结构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采样到蛋白质主链上来设计。我们称之为 结合位点趋同基序（COMBS）设计方法。 COMBS 用于设计 ABLER，第一个 配体结合蛋白从头开始设计，可与其目标配体（抗血栓药物阿哌沙班）结合 前所未有的高亲和力，无需对序列进行实验优化。 ABLER具有临床潜力 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 的结构在整体拓扑和预期分子方面都与设计模型一致 与配体的相互作用。该提案的目标 1 侧重于设计 ABLER 的变体以增加亲和力 并探索观察到的药物-蛋白质相互作用的分子基础。目标 2 重点关注水在 配体结合蛋白设计，由晶体中发现的水介导的蛋白质-配体相互作用驱动 结构。在此目标中，我将整理 PDB 中的水-蛋白质相互作用数据库，并使用这些数据库 在设计过程中对水介导的蛋白质-配体相互作用进行采样。我还将学习使用清水 分子动力学模拟来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（例如 磺基酪氨酸）进入哺乳动物细胞，因为迄今为止，实验室进化和文库筛选已经实现了这一目标 不成功。这些目标将增强我在分子生物学、计算蛋白质设计和 蛋白质结构表征（X 射线晶体学和 NMR）。本作品的 K99 部分位于 DeGrado 实验室将让我了解科学过程的各个方面，从开始到发表。比尔是一个世界 蛋白质设计专家，他的洞察力对于项目的成功至关重要。在UCSF，我将收获很多 我定期与伊森·韦斯 (Ethan Weiss) 会面，他带来了医学科学家的观点和 抗血栓研究和临床应用历史悠久。我与加州大学旧金山分校雷教授的合作 Wang 将使我了解非天然氨基酸掺入领域，这对于应用 COMBS 至关重要 寻找最有影响力的翻译后修饰模拟目标。拟议的研究和培训 本文将极大地补充我目前的技能和背景，这对我的研究至关重要 当我转变为独立首席研究员时。