Molecular Biotechnology, Institute of Molecular Biosciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
ACS Chem Biol. 2020 Apr 17;15(4):982-989. doi: 10.1021/acschembio.9b01022. Epub 2020 Mar 25.
Nonribosomal peptide synthetases (NRPSs) produce a wide variety of different natural products from amino acid precursors. In contrast to single protein NRPS, the NRPS of the bacterium producing the peptide-antimicrobial-Xenorhabdus (PAX) peptide consists of three individual proteins (PaxA/B/C), which interact with each other noncovalently in a linear fashion. The specific interactions between the three different proteins in this NRPS system are mediated by short C- and N-terminal docking domains (DDs). Here, we investigate the structural basis for the specific interaction between the DD from the protein PaxB and the DD from PaxC. The isolated DD peptides feature transient α-helical conformations in the absence of the respective DD partner. Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) titration experiments showed that the two isolated DDs bind to each other and form a structurally well-defined complex with a dissociation constant in the micromolar range as is typical for many DD interactions. Artificial linking of this DD pair via a flexible glycine-serine (GS) linker enabled us to solve the structure of the DD complex by NMR spectroscopy. In the complex, the two DDs interact with each other by forming a three helix bundle arranged in an overall coiled-coil motif. Key interacting residues were identified in mutagenesis experiments. Overall, our structure of the PaxB DD/PaxC DD complex represents an architecturally new type of DD interaction motif.
非核糖体肽合成酶（NRPSs）从氨基酸前体中产生各种各样的不同天然产物。与单个蛋白 NRPS 不同，产生肽抗生素 Xenorhabdus（PAX）肽的细菌的 NRPS 由三个单独的蛋白（PaxA/B/C）组成，它们以线性方式非共价相互作用。该 NRPS 系统中三个不同蛋白之间的特定相互作用由短的 C 端和 N 端对接结构域（DD）介导。在这里，我们研究了蛋白 PaxB 的 DD 与 PaxC 的 DD 之间特异性相互作用的结构基础。分离的 DD 肽在没有相应 DD 伴侣的情况下呈现瞬态 α-螺旋构象。等温滴定量热法（ITC）和核磁共振（NMR）滴定实验表明，两个分离的 DD 相互结合并形成结构上定义明确的复合物，解离常数在微摩尔范围内，这是许多 DD 相互作用的典型特征。通过柔性甘氨酸-丝氨酸（GS）接头将该 DD 对进行人工连接，使我们能够通过 NMR 光谱解决 DD 复合物的结构。在复合物中，两个 DD 通过形成三螺旋束相互作用，该三螺旋束以整体卷曲螺旋基序排列。突变实验确定了关键的相互作用残基。总的来说，我们的 PaxB DD/PaxC DD 复合物结构代表了一种新的 DD 相互作用基序。
