Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen–Deuterium Exchange Mass Spectrometry
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".