Partially Modified Peptide Intermediates in Lanthipeptide Biosynthesis Alter the Structure and Dynamics of a Lanthipeptide Synthetase

Lanthipeptide synthetases construct macrocyclic peptide natural products by catalyzing an iterative cascade of post-translational modifications. Class II lanthipeptide synthetases (LanM enzymes) catalyze multiple rounds of peptide dehydration and thioether macrocycle formation in a manner that guides precursor peptide maturation to the biologically active final product with high fidelity. The mechanistic details underlying the contradictory phenomena of substrate flexibility coupled with high biosynthetic fidelity have proven challenging to illuminate. In this work, we employ mass spectrometry to investigate how the structure of a maturing precursor lanthipeptide (HalA2) influences the local and global structure of its cognate lanthipeptide synthetase (HalM2). Using enzymatically synthesized HalA2 peptides that contain sets of native thioether macrocycles, we employ ion mobility mass spectrometry (IM-MS) to show that HalA2 macrocyclization alters the conformational landscape of the HalM2 enzyme in a systematic manner. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) studies show that local HalM2 structural dynamics also change in response to HalA2 post-translational modification. Notably, deuterium uptake in a critical HalM2 α-helical region depends on the number of thioether macrocycles present in the HalA2 core peptide. Binding of the isolated leader and core peptide portions of the modular HalA2 precursor led to a synergistic structuring of this α-helical region, providing evidence for distinct leader and core peptide binding sites that independently alter the dynamics of this functionally critical α-helix. The data support a mechanistic model where the sequential post-translational modification of HalA2 alters the conformational dynamics of HalM2 in regions of the enzyme that are known to be functionally critical.羊毛硫肽合成酶（Lanthipeptide synthetases）通过催化迭代级联的翻译后修饰反应，构建大环肽类天然产物。II型羊毛硫肽合成酶（LanM酶）可催化多轮肽脱水与硫醚大环形成反应，以高保真度指导前体肽成熟为具有生物活性的终产物。底物灵活性与高生物合成保真度并存这一矛盾现象背后的机制细节，长期以来难以阐明。 本研究利用质谱技术，探究成熟中的羊毛硫肽前体（HalA2）的结构如何影响其同源羊毛硫肽合成酶（HalM2）的局部与全局构象。 本研究使用酶法合成的带有天然硫醚大环结构的HalA2肽，借助离子迁移质谱（ion mobility mass spectrometry，IM-MS）证实，HalA2的大环化过程会系统性地改变HalM2酶的构象分布。 氢氘交换质谱（Hydrogen–deuterium exchange mass spectrometry，HDX-MS）研究显示，HalM2的局部结构动力学也会因HalA2的翻译后修饰而发生改变。值得注意的是，HalM2一个关键α螺旋区域的氘摄取量，取决于HalA2核心肽中硫醚大环的数量。 对模块化HalA2前体中分离得到的引导肽与核心肽部分进行结合实验，结果显示该α螺旋区域会出现协同构象变化，这为存在独立的引导肽与核心肽结合位点提供了证据——这两类位点可分别改变这一功能关键α螺旋的动力学特性。 本研究数据支持如下机制模型：HalA2的依次翻译后修饰，会改变HalM2酶中已知具有关键功能的区域的构象动力学。