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

基本信息

批准号：
10569523
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10569523
关键词：
4&apos -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 Dimensions 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 Productivity Protein Dynamics Proteins Regulation Research Role Sirolimus Site Specificity Structure Substrate Interaction 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 molecular dynamics novel pathogen peptide synthase preservation response tool

项目摘要

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。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SARA: a software environment for the analysis of relaxation data acquired with accordion spectroscopy.

DOI：
10.1007/s10858-013-9807-x
发表时间：
2014-02
期刊：
Journal of biomolecular NMR
影响因子：
2.7
作者：
Harden BJ;Frueh DP
通讯作者：
Frueh DP

NMR as a readout to monitor and restore the integrity of complex chemoenzymatic reactions.

DOI：
10.1016/j.jmr.2022.107265
发表时间：
2022-09
期刊：
JOURNAL OF MAGNETIC RESONANCE
影响因子：
2.2
作者：
Marincin, Kenneth A.;Hwang, Yousang;Kengmana, Everett S.;Meyers, David J.;Frueh, Dominique P.
通讯作者：
Frueh, Dominique P.

Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance.

通过核磁共振探测负载底物的载体蛋白。