Biochemical and structural analysis of unusual docking domains in non-ribosomal peptide synthetases (NRPS)

非核糖体肽合成酶 (NRPS) 中异常对接域的生化和结构分析

• 批准号: 368772725
• 负责人: Professor Dr. Henning D. Mootz
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/368772725?language=en
• 关键词: Biochemical; structural; analysis; unusual; docking

Non-ribosomal peptide synthetases (NRPSs) are the protein templates and catalysts for the biosynthesis of bioactive peptides like daptomycin and cyclosporine, which are of outstanding importance as pharmaceuticals. NRPSs consist of modules, each containing catalytic domains for the incorporation of one amino acid. In bacterial NRPSs the modules are usually spread over more than one polypeptide chain. The intermolecular subunit interactions are crucial for an ordered peptide synthesis and are mediated by various types of small docking domains. This project focuses on the protein-protein-interaction at the interface between epimerization (E) and condensation (C) domains, which is mediated by the so-called COM-domains (communication-mediating). The structure and the specificity-determining amino acids in this region are not or not sufficiently understood, respectively. Since they are at least partially embedded within the large proteins they cannot be studied in isolation. In previous work, we used genetically encoded photo-crosslinkers to derive spatial constraints that supported a helix-hand model of the interaction interface. However, a detailed molecular understanding of the structure is still missing. At the end of the last funding period we succeeded in the first crystallization of a COM domain. Meanwhile, we could solve the crystal structure, which forms the basis for this proposal. In this project, we will focus on the structure-based identification of the specificity-determining residues, their site-directed mutagenesis for the rational re-programming of COM domains and on the possible architectural role of COM domains. Since COM domains are also found in fused form (cis-COM) between internal E and C domains we postulate for the first time a role in the correct spatial interplay of the catalytic domains. We will test if split COM domains (trans-COM) can be generated from cis-COM domains and thereby delineate a conceivable evolutionary development. A detailed structural and biochemical understanding of COM domains will for the first time sufficiently characterize these unusual docking domains. Deciphering the trans-COM domain interactions will be a very important contribution to the combinatorial engineering of NRPSs, in particular because the COM domains hold the key to the E domains, which introduce D-amino acids into the products as unique and important structural elements. We will investigate purified, recombinant proteins as COM-domain model systems and will apply protein crystallography, various biochemical and biophysical methods as well as modern carbene footprinting.

非核糖体肽合成酶(NRPS)是生物合成达托霉素、环孢菌素等生物活性多肽的蛋白质模板和催化剂，具有重要的药用价值。NRPS由模块组成，每个模块包含用于结合一种氨基酸的催化域。在细菌NRPS中，模块通常分布在一个以上的多肽链上。分子间的亚基相互作用是合成有序多肽的关键，由各种类型的小分子对接结构域介导。这个项目主要研究在下向异构化(E)和缩聚(C)结构域之间的蛋白质-蛋白质相互作用，这是由所谓的COM结构域(通信中介)介导的。这一区域的结构和决定特异性的氨基酸分别还没有或没有被充分了解。因为它们至少部分嵌入到大蛋白中，所以不能孤立地研究它们。在以前的工作中，我们使用遗传编码的光交联器来推导支持交互界面的螺旋-手模型的空间约束。然而，对该结构的详细分子理解仍然缺乏。在上一个资助期结束时，我们成功地实现了COM域的第一次结晶。同时，我们可以解决晶体结构，这构成了这一建议的基础。在这个项目中，我们将专注于基于结构的特异性决定残基的鉴定，它们的定点突变，以合理地重新编程COM结构域，以及COM结构域可能的结构作用。由于COM结构域也在内部E和C结构域之间以融合形式(顺式COM)被发现，我们首次假设在催化结构域的正确空间相互作用中发挥作用。我们将测试是否可以从顺-COM域生成拆分的COM域(TRANS-COM)，从而描绘出一种可想象的进化发展。对COM域的详细结构和生化理解将首次充分描述这些不寻常的对接域。破译跨COM结构域的相互作用将对NRPS的组合工程做出非常重要的贡献，特别是因为COM结构域是E结构域的关键，E结构域将D-氨基酸作为唯一和重要的结构元素引入产品中。我们将研究纯化的重组蛋白质作为COM结构域模型系统，并将应用蛋白质结晶学、各种生化和生物物理方法以及现代卡宾足迹。