De novo design of small-molecule-binding proteins

小分子结合蛋白的从头设计

基本信息

批准号：
10683406
负责人：
Nicholas Polizzi
金额：
$ 24.9万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10683406
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 Fibrinolytic Agents 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 Productivity Protein Engineering Protein Structure Databases Proteins Publications Recording of previous events Research 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 repository delivery vehicle design functional group infancy insight meetings model design molecular dynamics molecular recognition mutant novel phosphohistidine porphyrin a professor programs protein data bank protein folding protein function protein protein interaction pyrrolysine screening sensor skills small molecule success thrombotic tyrosine O-sulfate unnatural amino acids water sampling

项目摘要

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，并扩展了我们的知识。通过从头开始创建蛋白质来实现蛋白质折叠和功能。该提案旨在开发一种蛋白质。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配体结合蛋白设计的最新技术很大程度上依赖于随机实验。如果我们真正了解蛋白质如何结合小分子，我们应该能够进行优化和筛选。直接从计算机模型到紧密结合剂推动这一提议的假设是蛋白质使用a。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被用来揭示这组蛋白质。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。将通过将配体的所有功能基团采样到蛋白质主链上来设计。设计方法 COMBS 用于设计第一个 ABLER。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，无需实验优化序列即可具有临床潜力。作为抗凝血解毒剂的相关性，尽管这超出了高分辨率晶体的范围。 ABLER 的结构在整体拓扑和预期分子方面都与设计模型一致该提案的目标 1 侧重于设计 ABLER 的变体以增加亲和力。并探究观察到的药物-蛋白质相互作用的分子基础。目标 2 重点关注水在药物中的作用。配体结合蛋白设计，由晶体中发现的水介导的蛋白质-配体相互作用驱动在这个目标中，我将整理 PDB 中的水-蛋白质相互作用数据库，并使用它们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，我还将学习使用显式水。 Aim 3 使用 COMBS 进行分子动力学模拟来严格评估水在结合中的作用。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）。 DeGrado 实验室将让我了解科学过程的各个方面，从比尔是一个世界。蛋白质设计方面的专家，他的洞察力对于项目的成功至关重要，在加州大学旧金山分校，我将受益匪浅。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我与加州大学旧金山分校雷教授的合作有着悠久的抗血栓研究和临床应用历史。 Wang 将使我了解非天然氨基酸掺入领域，这对于应用 COMBS 至关重要拟定的研究和培训的最有影响力的目标。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。