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

非核糖体肽合成酶（Non-ribosomal peptide synthetases，NRPSs）是生物合成达托霉素（daptomycin）和环孢霉素（cyclosporine）等生物活性肽的蛋白质模板和催化剂。NRPS由模块组成，每个模块含有用于掺入一个氨基酸的催化结构域。在细菌NRPS中，模块通常分布在一条以上的多肽链上。分子间亚基相互作用对于有序的肽合成是至关重要的，并且由各种类型的小对接结构域介导。本项目的重点是蛋白质-蛋白质相互作用在差向异构化（E）和缩合（C）结构域之间的界面，这是由所谓的COM-结构域（通信介导）介导。该区域的结构和特异性决定氨基酸分别没有或没有充分理解。由于它们至少部分嵌入在大蛋白质中，因此无法孤立地进行研究。在以前的工作中，我们使用基因编码的光交联剂来获得空间约束，支持交互界面的螺旋手模型。然而，仍然缺少对结构的详细分子理解。在上一个融资期结束时，我们成功地实现了COM域的第一个结晶。同时，我们可以解决晶体结构，这构成了这个建议的基础。在这个项目中，我们将专注于基于结构的识别的特异性决定残基，其定点诱变COM域的合理重新编程和COM域的可能的建筑作用。由于COM结构域也被发现在内部E和C结构域之间的融合形式（顺式COM），我们假设第一次在正确的空间相互作用的催化结构域的作用。我们将测试，如果分裂COM域（trans-COM）可以从顺式COM域生成，从而描绘一个可以想象的进化发展。COM域的详细结构和生物化学的理解将首次充分表征这些不寻常的对接域。破译trans-COM结构域相互作用将对NRPS的组合工程做出非常重要的贡献，特别是因为COM结构域掌握着E结构域的关键，E结构域将D-氨基酸作为独特且重要的结构元件引入产物中。我们将研究纯化的重组蛋白作为COM结构域模型系统，并将应用蛋白质晶体学，各种生物化学和生物物理方法以及现代卡宾足迹。

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.