Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry
Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry
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DOI：
10.1021/jacs.9b06020
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发表时间：
2019-09-18
影响因子：
15
通讯作者：
Thibodeaux, Christopher J.
中科院分区：
文献类型：
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作者：
Habibi, Yeganeh;Uggowitzer, Kevin A.;Thibodeaux, Christopher J.
核糖体合成和翻译后修饰多肽(Ripps)的生物合成是通过基因编码的前体多肽的多步成熟进行的，这些前体多肽通常是由具有多功能和迭代活性的酶催化的。最近的研究表明，在其他因素中，酶：多肽复合体的构象采样可能在确定这些系统中的动力学以及最终的翻译后修饰方面发挥关键作用。然而，这些假定的构象采样机制的详细特征还不可能在许多RIPP生物合成系统上实现。在这项研究中，我们首次全面应用氢-氚交换质谱仪(HDX-MS)来研究RIPP生物合成酶的生物物理性质。以已知的第二类羊毛肽合成酶HalM2为模型系统，我们用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，我们已经确定了新的前体多肽结合元件，发现了由配体结合和ATP水解触发的跨酶的长距离结构通讯，并检测到HalM2合成酶与模块化HalA2前体多肽底物的前导和核心肽亚域之间的特异性相互作用。本研究中发现的动态HalM2元件的功能相关性通过生化分析和一组HDX-MS引导的变异酶的动力学分析来验证。总体而言，这些数据提供了大量关于LAMM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合理操作和工程设计提供信息。此外，这项工作突出了HDX-MS平台在揭示重要的生物物理性质和酶结构动力学方面的广泛用途，这些性质和酶结构动力学可能在RIPP的生物合成中发挥广泛的作用。
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.