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: 10358654
• 负责人: Dominique Pascal Frueh
• 金额: $ 34.39万
• 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/10358654
• 关键词: 4' -phosphopantetheine; Active Sites; Anabolism; Antibiotics; Antineoplastic Agents; Architecture; Bacitracin; Binding; Binding Sites; Biological; Biological Assay; Bleomycin; Catalytic Domain; Chemicals; Cholera; Communication; Complex; Computing Methodologies; Coupled; Couples; Coupling; Crystallography; Cyclization; Development; Docking; Drug Design; Engineering; Enzyme Kinetics; Enzymes; Escherichia coli; Gene Activation; Immunosuppressive Agents; Ligand Binding; Ligand Binding Domain; Methodological Studies; Methods; Modification; Molecular; Mutation; Mycobacterium tuberculosis; Names; Natural Products; Nuclear Magnetic Resonance; Pharmacologic Substance; Physical condensation; Plague; Positioning Attribute; Post-Translational Protein Processing; Proteins; Regulation; Research; Role; Sirolimus; Site; Specificity; Structure; Substrate Specificity; System; Techniques; Tertiary Protein Structure; Therapeutic; Tuberculosis; Urinary tract infection; Uropathogenic E. coli; Vibrio cholerae; Virulence; Yersinia pestis; antitumor agent; arm; enzyme mechanism; improved; interest; intermolecular interaction; kinetic model; macromolecule; microbial; novel; pathogen; peptide synthase; preservation; response; tool

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.

从基因激活到酶调节的生物活性是通过分子相互作用发生的， 其调节可以描述为通过化学作用重新分配分子间相互作用 修饰或配体结合。不幸的是，当蛋白质通过远程与两个伙伴相互作用时 结合位点，可以解释蛋白质内部变化如何改变通讯的分子机制 蛋白质之间的联系往往是难以捉摸的。这一挑战限制了设计可以改变相互作用以进行救援的药物 异常的生物活性。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组装简单的 以装配线方式将基材加工成复杂的产品。这些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（博莱霉素）和免疫抑制剂（雷帕霉素），但是 其他赋予病原体（大肠杆菌、霍乱弧菌、鼠疫耶尔森氏菌）毒力。 NRPS 是人们广泛关注的焦点 因为对它们进行改造以纳入不同的底物可以生产新型药物。然而， 就像工厂的装配线一样，NRPS 不是静态的，它们的域在动态中瞬时交互 建筑学。因此，了解 NRPS 的分子机制并对其进行潜在的改造至关重要 相当于解决一个动态的、多维的难题。值得注意的是，目前尚不清楚底物如何与 一些域，以及这些交互如何反过来促进多个合作伙伴域之间的通信， 这就是我们上面描述的蛋白质的情况。我们发现域内的结构动力学 响应底物以促进域之间的相互作用，并且它们耦合远程结合位点并 酶活性位点。也就是说，动力学包含理解底物识别和远程的关键 沟通。该提案旨在提供关键 NRPS 内动力学的分子描述 域并揭示其在底物和伙伴域识别中的功能。我们将使用核磁 共振，可以在原子水平上描述实验动力学，以描述动态响应 当结构域相互作用时，以及与合成过程中的底物相互作用时。这些研究是 补充了功能测定、计算方法和晶体学，并将回答 关于蛋白质通讯、酶机制和远程通讯的长期问题 蛋白质。该结果将为将外源底物识别设计成 NRPS 提供基础，这是一种条件 通过 NRPS 重编程生产新药物。