McGill University , Department of Chemistry , 801 Sherbrooke Street , West Montréal , Québec , Canada H3A 0B8.
J Am Chem Soc. 2019 Sep 18;141(37):14661-14672. doi: 10.1021/jacs.9b06020. Epub 2019 Sep 6.
The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) proceeds via the multistep maturation of genetically encoded precursor peptides, often catalyzed by enzymes with multiple functions and iterative activities. Recent studies have suggested that, among other factors, conformational sampling of enzyme:peptide complexes likely plays a critical role in defining the kinetics and, ultimately, the set of post-translational modifications in these systems. However, detailed characterizations of these putative conformational sampling mechanisms have not yet been possible on many RiPP biosynthetic systems. In this study, we report the first comprehensive application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study the biophysical properties of a RiPP biosynthetic enzyme. Using the well-characterized class II lanthipeptide synthetase HalM2 as a model system, we have employed HDX-MS to demonstrate that HalM2 is indeed a highly structurally dynamic enzyme. Using this HDX-MS approach, we have identified novel precursor peptide binding elements, have uncovered long-range structural communication across the enzyme that is triggered by ligand binding and ATP hydrolysis, and have detected specific interactions between the HalM2 synthetase and the leader- and core-peptide subdomains of the modular HalA2 precursor peptide substrate. The functional relevance of the dynamic HalM2 elements discovered in this study are validated with biochemical assays and kinetic analysis of a panel of HDX-MS guided variant enzymes. Overall, the data have provided a wealth of fundamentally new information on LanM systems that will inform the rational manipulation and engineering of these impressive multifunctional catalysts. Moreover, this work highlights the broad utility of the HDX-MS platform for revealing important biophysical properties and enzyme structural dynamics that likely play a widespread role in RiPP biosynthesis.
核糖体合成和翻译后修饰肽（RiPPs）的生物合成是通过遗传编码前体肽的多步成熟进行的，通常由具有多种功能和迭代活性的酶催化。最近的研究表明，除其他因素外，酶：肽复合物的构象采样可能在定义这些系统中的动力学和最终翻译后修饰方面起着关键作用。然而，在许多 RiPP 生物合成系统中，尚未能够详细描述这些假定的构象采样机制。在这项研究中，我们报告了首次全面应用氢氘交换质谱（HDX-MS）来研究 RiPP 生物合成酶的生物物理性质。使用经过充分表征的 II 类短杆菌肽合成酶 HalM2 作为模型系统，我们已经使用 HDX-MS 证明了 HalM2 确实是一种高度结构动态的酶。使用这种 HDX-MS 方法，我们确定了新的前体肽结合元件，揭示了配体结合和 ATP 水解触发的酶的远程结构通讯，并检测到 HalM2 合成酶与模块化 HalA2 前体肽底物的引导肽和核心肽亚结构域之间的特定相互作用。在这项研究中发现的动态 HalM2 元件的功能相关性通过生化测定和一组 HDX-MS 指导的变体酶的动力学分析得到了验证。总体而言，这些数据提供了大量关于 LanM 系统的全新信息，这些信息将为这些令人印象深刻的多功能催化剂的理性操作和工程提供信息。此外，这项工作强调了 HDX-MS 平台在揭示可能在 RiPP 生物合成中广泛发挥作用的重要生物物理性质和酶结构动力学方面的广泛应用。
