LAUSR

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.