Exploring the Heterogeneous Structural Dynamics of Class II Lanthipeptide Synthetases with Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS)

Class II lanthipeptide synthetases (LanM enzymes) catalyze
the
installation of multiple thioether bridges into genetically encoded
peptides to produce macrocyclic lanthipeptides, a class of biologically
active natural products. Collectively, LanM enzymes install thioether
rings of different sizes, topologies, and stereochemistry into a vast
array of different LanA precursor peptide sequences. The factors that
govern the outcome of the LanM-catalyzed reaction cascade are not
fully characterized but are thought to involve both intermolecular
interactions and intramolecular conformational changes in the [LanM:LanA]
Michaelis complex. To test this hypothesis, we have combined AlphaFold
modeling with hydrogen–deuterium exchange mass spectrometry
(HDX-MS) analysis of a small collection of divergent LanM/LanA systems
to investigate the similarities and differences in their conformational
dynamic properties. Our data indicate that LanA precursor peptide
binding triggers relatively conserved changes in the structural dynamics
of the LanM dehydratase domain, supporting the existence of a similar
leader peptide binding mode across the LanM family. In contrast, changes
induced in the dynamics of the LanM cyclase domain were more highly
variable between enzymes, perhaps reflecting different peptide–cyclase
interactions and/or different modes of allosteric activation in class
II lanthipeptide biosynthesis. Our analysis highlights the ability
of the emerging AlphaFold platform to predict protein–peptide
interactions that are supported by other lines of experimental evidence.
The combination of AlphaFold modeling with HDX-MS analysis should
emerge as a useful approach for investigating other conformationally
dynamic enzymes involved in peptide natural product biosynthesis.