Department of Chemistry and Centre de Recherche en Biologie Structurale, McGill University, 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada.
Biochemistry. 2021 May 18;60(19):1506-1519. doi: 10.1021/acs.biochem.1c00085. Epub 2021 Apr 22.
Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These genetically encoded peptides are biosynthesized by multifunctional enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity and catalyze iterative rounds of post-translational modification. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native mass spectrometry coupled to ion mobility (native IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its noncovalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes native IM-MS as a versatile approach for characterizing intermolecular interactions and for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.
类硫醚抗生素是核糖体合成和翻译后修饰的肽（RiPP）天然产物。这些基因编码的肽由多功能酶（类硫醚抗生素合成酶）生物合成，这些酶具有宽松的底物特异性，并催化翻译后修饰的迭代循环。最近的证据表明，一些类硫醚抗生素合成酶是结构动态的酶，它们通过前体肽结合被别构激活，并且酶-肽复合物的构象采样可能在定义生物合成事件的效率和序列方面发挥重要作用。这些“生物物理”过程对于定义合成酶的活性和功能至关重要，但用现有的方法学研究仍然极具挑战性。在此，我们表明，与离子淌度（native IM-MS）联用的天然质谱提供了一种强大而敏感的方法，可用于研究类硫醚抗生素合成酶的构象景观和分子间相互作用。也就是说，我们证明了 II 类类硫醚抗生素合成酶（HalM2）及其与同源 HalA2 前体肽的非共价复合物可以以保留天然结构和分子间酶-肽接触的方式递送至气相中。此外，对天然折叠离子的气相离子淌度研究表明，肽结合和对 HalM2 动态结构元件的突变改变了酶的构象景观。总而言之，这些数据支持了先前的观点，即类硫醚抗生素合成酶是结构动态的酶，它们会响应前体肽结合而发生与功能相关的构象变化。这项工作确立了 native IM-MS 作为一种通用方法，用于表征分子间相互作用，并揭示 RiPP 生物合成系统中蛋白质结构与生化功能之间的关系。
