Partially Modified Peptide Intermediates in Lanthipeptide Biosynthesis Alter the Structure and Dynamics of a Lanthipeptide Synthetase

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.