Lanthipeptide synthetases construct macrocyclic peptide natural products by catalyzing an iterative cascade of post-translational modifications. Class II lanthipeptide synthetases (LanM enzymes) catalyze multiple rounds of peptide dehydration and thioether macrocycle formation in a manner that guides precursor peptide maturation to the biologically active final product with high fidelity. The mechanistic details underlying the contradictory phenomena of substrate flexibility coupled with high biosynthetic fidelity have proven challenging to illuminate. In this work, we employ mass spectrometry to investigate how the structure of a maturing precursor lanthipeptide (HalA2) influences the local and global structure of its cognate lanthipeptide synthetase (HalM2). Using enzymatically synthesized HalA2 peptides that contain sets of native thioether macrocycles, we employ ion mobility mass spectrometry (IM-MS) to show that HalA2 macrocyclization alters the conformational landscape of the HalM2 enzyme in a systematic manner. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that local HalM2 structural dynamics also change in response to HalA2 post-translational modification. Notably, deuterium uptake in a critical HalM2 α-helical region depends on the number of thioether macrocycles present in the HalA2 core peptide. Binding of the isolated leader and core peptide portions of the modular HalA2 precursor led to a synergistic structuring of this α-helical region, providing evidence for distinct leader and core peptide binding sites that independently alter the dynamics of this functionally critical α-helix. The data support a mechanistic model where the sequential post-translational modification of HalA2 alters the conformational dynamics of HalM2 in regions of the enzyme that are known to be functionally critical.