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.

项目概要 从基因激活到酶调节的生物活性通过分子发生 相互作用及其调节可以描述为分子间相互作用的重新分配 通过化学修饰或配体结合。不幸的是，当蛋白质与 两个伙伴通过远程结合位点，分子机制可以解释如何 蛋白质内部的变化会改变蛋白质之间的通讯，这通常是难以捉摸的。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 以装配线方式将简单的基材组装成复杂的产品。产品 通常是有价值的治疗药物，包括抗生素（杆菌肽）、抗肿瘤药物 （博莱霉素）和免疫抑制剂（雷帕霉素），但其他药物赋予病原体毒力 （大肠杆菌、霍乱弧菌、鼠疫耶尔森氏菌）。 NRPS 是人们广泛关注的焦点，因为工程 它们结合不同的底物可以生产新型药物。然而，就像 工厂的装配线、NRPS 不是静态的，它们的域在瞬态中相互作用 动态架构。因此，了解 NRPS 的分子机制，以及 对它们进行潜在的设计，无异于解决一个动态的、多维的难题。 值得注意的是，目前尚不清楚底物如何与某些域相互作用，以及这些相互作用如何， 反过来，促进几个合作伙伴域之间的沟通，这就是我们的情况 上面对蛋白质进行了描述。我们发现域内的结构动力学响应 促进域之间相互作用的底物，并且它们耦合远程结合位点 和酶活性位点。也就是说，动力学包含理解两种底物的关键 识别和远程通​​信。该提案旨在提供分子描述 关键 NRPS 域内的动态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 在原子水平上进行实验动力学，以描述域时的动态响应 相互作用，以及与合成过程中的底物相互作用。这些研究是 辅以功能测定、计算方法和晶体学，并将 回答有关蛋白质通讯、酶机制和远程控制等长期存在的问题 蛋白质内部的通讯。研究结果将为外源底物的设计提供依据 获得 NRPS 认可，这是通过 NRPS 生产新药品的条件 重新编程。