Dynamic allosteric communication within nonribosomal peptide synthetase cyclization domains

非核糖体肽合成酶环化域内的动态变构通讯

• 批准号: 10387089
• 负责人: Dominique Pascal Frueh
• 金额: $ 10.43万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387089
• 关键词: Active Sites; Anabolism; Antibiotics; Antineoplastic Agents; Architecture; Bacitracin; Binding; Binding Sites; Biological; Biological Assay; Bleomycin; Chemicals; Cholera; Communication; Complex; Computing Methodologies; Crystallography; Cyclization; Drug Design; Engineering; Enzymes; Escherichia coli; Gene Activation; Immunosuppressive Agents; Ligand Binding; Modification; Molecular; Mycobacterium tuberculosis; Natural Products; Nuclear Magnetic Resonance; Pharmacologic Substance; Plague; Proteins; Regulation; Research; Sirolimus; Structure; System; Tertiary Protein Structure; Therapeutic; Tuberculosis; Urinary tract infection; Uropathogenic E. coli; Vibrio cholerae; Virulence; Yersinia pestis; antitumor agent; enzyme mechanism; improved; interest; intermolecular interaction; microbial; novel; pathogen; peptide synthase; response

Project Summary Biological activity, ranging from gene activation to enzyme regulation, occurs through molecular interactions, and its regulation can be described as a redistribution of intermolecular interactions through chemical modifications or ligand binding. Unfortunately, when a protein interacts with two partners through remote binding sites, molecular mechanisms that would explain how changes within proteins alter the communication between proteins are often elusive. This challenge limits designing drugs that could alter interactions to rescue abnormal biological activity. The conundrum also applies to microbial enzymatic factories called nonribosomal peptide synthetases (NRPSs). NRPSs use contiguous protein domains to incorporate and assemble simple substrates into complex products in an assembly line fashion. The products are often valuable therapeutics, including antibiotics (bacitracin), antitumor agents (bleomycin), and immunosuppressants (rapamycin), but others confer virulence to pathogens (E. coli, V. cholerae, Y. pestis). NRPSs are the focus of much interest because engineering them to incorporate different substrates could produce novel pharmaceuticals. However, like assembly lines in factories, NRPSs are not static, and their domains interact transiently in a dynamic architecture. Thus, understanding the molecular mechanisms of NRPSs, and potentially engineering them, is tantamount to solving a dynamic, multi-dimensional puzzle. Notably, it is unknown how substrates interact with some domains, and how these interactions, in turn, promote communication between several partner domains, which is the situation we described above for proteins. We found that structural dynamics within domains respond to substrates to promote interactions between domains and that they couple remote binding sites and enzymatic active sites. That is, dynamics contain keys to understanding both substrate recognition and remote communication. This proposal aims to provide a molecular description of the dynamics within critical NRPS domains and reveal its function in substrate and partner domain recognition. We will use nuclear magnetic resonance, which can describe experimentally dynamics at the atomic-level, to describe dynamic responses when domains interact with each other, and with substrates as they do during synthesis. The studies are supplemented with functional assays, computational methods, and crystallography, and will answer longstanding questions about protein communication, enzyme mechanisms, and remote communication within proteins. The results will provide a basis to engineer exogenous substrate recognition into NRPSs, a condition for producing new pharmaceuticals through NRPS reprogramming.

项目摘要 生物活性，从基因激活到酶调节，通过分子生物学途径发生。 相互作用，其调节可以被描述为分子间相互作用的重新分布 通过化学修饰或配体结合。不幸的是，当蛋白质与 两个伴侣通过远程结合位点，分子机制，可以解释如何 蛋白质内的变化改变蛋白质之间的通讯往往是难以捉摸的。这 挑战限制了设计可以改变相互作用以拯救异常生物学的药物 活动这个难题也适用于称为非核糖体的微生物酶工厂 肽合成酶（NRPS）。NRPS使用连续的蛋白质结构域来整合和 以装配线的方式将简单的基板装配成复杂的产品。产品 通常是有价值的治疗剂，包括抗生素（杆菌肽）、抗肿瘤剂 （博来霉素）和免疫抑制剂（雷帕霉素），但其他赋予病原体的毒力 （E. coli、霍乱弧菌、Y.鼠疫）。NRPS是非常感兴趣的焦点，因为工程 它们结合不同的底物可以产生新的药物。但是像 在工厂的装配线上，NRPS不是静态的，它们的结构域以一种短暂的方式相互作用。 动态架构因此，了解NRPS的分子机制， 潜在地设计它们，就等于解决一个动态的、多维的难题。 值得注意的是，底物如何与某些结构域相互作用，以及这些相互作用如何， 反过来，促进几个合作伙伴领域之间的沟通，这是我们的情况， 上面提到的蛋白质。我们发现，结构域内的结构动力学响应于 底物，以促进结构域之间的相互作用，并且它们偶联远程结合位点 和酶活性位点。也就是说，动力学包含理解两种基质的关键 识别和远程通信。该提案旨在提供一种分子描述， 关键NRPS结构域内的动力学，并揭示其在底物和伴侣中的功能 域识别我们将使用核磁共振，它可以描述 实验动力学在原子水平上，描述动态响应时，域 它们在合成过程中相互作用，并与底物相互作用。这些研究 补充功能测定，计算方法和晶体学，并将 回答关于蛋白质通讯，酶机制和远程 蛋白质内的通讯研究结果为外源底物的工程改造提供了依据 识别NRPS，通过NRPS生产新药的条件 重新编程