Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis
Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis
复制标题
DOI：
10.1016/j.chembiol.2009.02.009
复制
发表时间：
2009-04-24
影响因子：
--
通讯作者：
Burkart, Michael D.
中科院分区：
文献类型：
--
作者：
Hur, Gene H.;Meier, Jordan L.;Burkart, Michael D.
非核糖体肽合成酶（NRPS）蛋白之间的选择性蛋白质-蛋白质相互作用由通讯介导（COM）结构域控制，负责生物合成中间体的适当移位以产生天然产物。在这项研究中，我们开发了一种交联试验，利用生物正交探针与载体蛋白修饰兼容，用于探测NRPS酶COM结构域之间的蛋白质相互作用。采用Huisgen 1，3-偶极环加成的叠氮化物和炔，我们研究了在tyrocidine途径内的同源NRPS模块的交联，并证明了我们的面板对相容COM结构域的选择性蛋白质相互作用的交联探针的灵敏度。这些研究表明，无铜交联底物独特地提供了蛋白质-蛋白质相互作用的诊断探针。同样，这些交联探针作为理想的化学工具，NRPS模块之间的结构研究，其中缺乏功能测定。
Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.