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学活性通过分子相互作用发生， 它的调节可以描述为通过化学的分子间相互作用的重新分布 修饰或配体结合。不幸的是，当蛋白质通过远程与两个伴侣相互作用时 结合位点，分子机制，这些机制可以解释蛋白质内的变化如何改变通信 蛋白质之间通常难以捉摸。这项挑战限制了设计可能改变相互作用以营救的药物的限制 异常生物学活性。该难题还适用于称为非透射体的微生物酶促工厂 肽合酶（NRPS）。 NRPS使用连续的蛋白质结构域来合并和组装简单 以装配线方式构成复杂产品。这些产品通常是有价值的治疗方法， 包括抗生素（杆菌蛋白），抗肿瘤剂（博来霉素）和免疫抑制剂（雷帕霉素），但 其他人则将毒力赋予病原体（E. Coli，V。Cholerae，Y。Pestis）。 NRPS是引起人们关注的重点 因为使它们融合不同的底物可以生产新的药物。然而， 像工厂中的装配线一样，NRPS不是静态的，它们的域在动态上瞬时相互作用 建筑学。因此，了解NRPS的分子机制以及潜在的工程，是 解决动态的多维难题的牵头。值得注意的是，底物如何与 一些域，以及这些互动如何促进几个合作伙伴域之间的沟通， 这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互动，并且它们将远程绑定站点与 酶促活性位点。也就是说，动态包含了解底物识别和远程的密钥 沟通。该建议旨在提供对关键NRP中动力学的分子描述 域并揭示其在底物和合作伙伴域识别中的功能。我们将使用核磁 共振可以描述原子级的实验动力学，以描述动态响应 当域相互相互作用时，像合成过程中一样与底物相互作用。研究是 补充功能测定，计算方法和晶体学，并将回答 关于蛋白质通信，酶机制和远程通信的长期问题 蛋白质。结果将为工程师的外源底物识别提供基础， 用于通过NRP重新编程生产新的药物。