Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

Abstract

Lanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. 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 nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-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 non-covalent 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, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. 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 nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.