Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen–Deuterium Exchange Mass Spectrometry

The
biosynthesis of ribosomally synthesized and post-translationally
modified peptides (RiPPs) proceeds via the multistep maturation of
genetically encoded precursor peptides, often catalyzed by enzymes
with multiple functions and iterative activities. Recent studies have
suggested that, among other factors, conformational sampling of enzyme:peptide
complexes likely plays a critical role in defining the kinetics and,
ultimately, the set of post-translational modifications in these systems.
However, detailed characterizations of these putative conformational
sampling mechanisms have not yet been possible on many RiPP biosynthetic
systems. In this study, we report the first comprehensive application
of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to
study the biophysical properties of a RiPP biosynthetic enzyme. Using
the well-characterized class II lanthipeptide synthetase HalM2 as
a model system, we have employed HDX-MS to demonstrate that HalM2
is indeed a highly structurally dynamic enzyme. Using this HDX-MS
approach, we have identified novel precursor peptide binding elements,
have uncovered long-range structural communication across the enzyme
that is triggered by ligand binding and ATP hydrolysis, and have detected
specific interactions between the HalM2 synthetase and the leader-
and core-peptide subdomains of the modular HalA2 precursor peptide
substrate. The functional relevance of the dynamic HalM2 elements
discovered in this study are validated with biochemical assays and
kinetic analysis of a panel of HDX-MS guided variant enzymes. Overall,
the data have provided a wealth of fundamentally new information on
LanM systems that will inform the rational manipulation and engineering
of these impressive multifunctional catalysts. Moreover, this work
highlights the broad utility of the HDX-MS platform for revealing
important biophysical properties and enzyme structural dynamics that
likely play a widespread role in RiPP biosynthesis.