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，都是通过分子发生的 相互作用及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，当一种蛋白质与 两个伙伴通过远程结合位点，分子机制可以解释 蛋白质内部的变化改变了蛋白质之间的通讯，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挽救异常生物的药物 活动。这个难题也适用于被称为非核糖体的微生物酶工厂 多肽合成酶(NRPS)。NRPS使用连续的蛋白质结构域来整合和 以流水线的方式将简单的基板组装成复杂的产品。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)，但其他药物则赋予病原体毒力 (大肠杆菌、霍乱弧菌、鼠疫耶尔森氏菌)。NRPS是人们非常感兴趣的焦点，因为工程 它们将不同的底物结合在一起，可以生产出新型药物。然而，就像 工厂中的装配线，NRPS不是静态的，它们的域在 动态架构。因此，了解NRPS的分子机制，以及 对它们进行潜在的工程设计，相当于解决了一个动态的、多维的谜题。 值得注意的是，目前尚不清楚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交流，这就是我们的情况 上面针对蛋白质进行了描述。我们发现，域内的结构动力学响应于 促进结构域之间相互作用的底物，以及它们耦合远程结合位点的底物 和酶活性部位。也就是说，动力学包含了理解这两种底物的关键 识别和远程通信。这项提案旨在提供一种分子描述 NRPS关键结构域内的动力学及其在底物和配对中的功能 域识别。我们将使用核磁共振，它可以描述 原子级别的实验动力学，以描述域时的动态响应 相互作用，并与底物相互作用，就像它们在合成过程中所做的那样。这些研究是 补充了功能分析、计算方法和结晶学，并将 回答长期存在的有关蛋白质通讯、酶机制和远程的问题 蛋白质内部的交流。研究结果将为外源底物工程提供依据。 认识NRPS，是通过NRPS生产新药的条件 重新编程。