Exploring the Conformational Landscape of a Lanthipeptide Synthetase Using Native Mass Spectrometry

Abstract

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These genetically encoded peptides are biosynthesized by multifunctional enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity and catalyze iterative rounds of posttranslational modification. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding and that conformational sampling of the enzyme−peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native mass spectrometry coupled to ion mobility (native IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its noncovalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme−peptide contacts. Moreover, gas phase ion mobility studies of the natively folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes native IM-MS as a versatile approach for characterizing intermolecular interactions and for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

Mutations in Dynamic Structural Elements Alter the Kinetics and Fidelity of the Multifunctional Class II Lanthipeptide Synthetase, HalM2

“…On the basis of these observations, we speculate that the interaction of these elements defines a HalA2 binding site at the interface of the F331–Q340 helix and cyclase β-sheet that somehow facilitates the preferred cyclization order (B → A → C/D) and the strong coupling of the third and fourth cyclizations observed in wt HalM2, HalM2 P405A , HalM2 T386A/D387A , and HalM2 P349A . This interpretation is consistent with (i) recent photochemical cross-linking studies suggesting a direct interaction between the HalA2 leader peptide and the cyclase domain β-sheet, (ii) the observation that HalA2 binding decreases the extent of deuterium exchange in the β-sheet and F331–Q340 helix, and (iii) the recent observation from native mass spectrometry studies that HalM2 can bind 2 equiv of HalA2 …”

“…HDX-MS analysis of the mutant enzymes in the presence and absence of ligands shows that the structural dynamics in many of the elements targeted for mutation are coupled, despite these elements being distant from each other in the HalM2 homology model. Finally, structural and mechanistic interpretations derived from the HDX-MS and kinetic data are bolstered by molecular dynamics simulations, which provide evidence for direct contact between the P349–P405 loop and the N-terminal helical capping domain involved in HalA2 leader peptide binding. , These studies build on recent observations suggesting that LanMs exhibit complex dynamic properties that are intimately tied to their structures and functions. , More generally, this study illustrates how a combination of mass spectrometry-based approaches can provide unique levels of functional insight into the complex, multistep mechanisms of lanthipeptide synthetases and other RiPP biosynthetic enzymes.…”

“…22,33 These studies build on recent observations suggesting that LanMs exhibit complex dynamic properties that are intimately tied to their structures and functions. 22,37 More generally, this study illustrates how a combination of mass spectrometry-based approaches can provide unique levels of functional insight into the complex, multistep mechanisms of lanthipeptide synthetases and other RiPP biosynthetic enzymes.…”

“…This interpretation is consistent with (i) recent photochemical cross-linking studies suggesting a direct interaction between the HalA2 leader peptide and the cyclase domain β-sheet, 33 (ii) the observation that HalA2 binding decreases the extent of deuterium exchange in the β-sheet and F331−Q340 helix, and (iii) the recent observation from native mass spectrometry studies that HalM2 can bind 2 equiv of HalA2. 37 Strikingly, despite the perturbations to the preferred cyclization sequences in many of the HalM2 variants, each enzyme converts a substantial portion of the HalA2 material into fully modified products that possess the correct thioether topology (compare F [prod] values in Table 2). This observation is in line with studies of other mutated and engineered LanM/ LanA systems.…”

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

“…22−24 These data suggest that different LanM−LanA binding modes may exist, such that the LanA core peptide can be presented to the LanM active sites from different orientations depending on the enzyme system. The high amino acid sequence divergence of LanA peptides 25 and the recent observation of multiple LanA binding sites on some LanMs 3,26,27 seem to support this possibility. The hypothesis that emerges from these biochemical studies is that while the chemistry of thioether bridge formation in LanM/LanA systems is highly conserved, 28−30 the precise sequence of biosynthetic transformations might be guided by more subtle enzyme−peptide interactions.…”