Insights into the Dynamic Structural Properties of a Lanthipeptide

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Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry Yeganeh Habibi, Kevin Adam Uggowitzer, Hassan Issak, and Christopher J. Thibodeaux J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06020 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Journal of the American Chemical Society

Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry Yeganeh Habibi, Kevin A. Uggowitzer, Hassan Issak, and Christopher J. Thibodeaux* McGill University, Department of Chemistry 801 Sherbrooke St. West Montréal, Québec, Canada, H3A 0B8 *To whom correspondence should be addressed Phone: 1-(514)-398-3637 Email: [email protected]

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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 characterization 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 has 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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Introduction Lanthipeptide synthetases are an interesting group of ribosomally-synthesized and posttranslationally modified peptide (RiPP) natural product biosynthetic enzymes that catalyze the formation of thioether rings in genetically encoded precursor peptide substrates to make products called lanthipeptides, which often function as antimicrobial compounds.1 The biosynthesis of all lanthipeptides involves the dehydration of serine and threonine residues in a precursor peptide (LanA), followed by intramolecular Michael-type addition of cysteine thiols onto the nascent dehydration sites to generate thioether crosslinks (Figure 1A).

Like the vast majority of RiPPs, lanthipeptide precursors are

structurally modular and are comprised of an N-terminal leader peptide (for biosynthetic enzyme recognition and cellular export) and a C-terminal core peptide which harbors the post-translational modification sites.

Most lanthipeptides contain multiple thioether rings, which necessitates a

synthetase with relaxed substrate specificity that functions iteratively on the maturing peptide to complete the multistep biosynthesis. The molecular mechanisms by which these fascinating enzymes recognize their substrates and coordinate their multiple activities in order to generate specific final products, or in some cases a range of final products,2 are not fully understood.

Detailed

characterization of these mechanisms will greatly expand the potential biotechnological applications of lanthipeptide synthetases, as well as other RiPP biosynthetic enzymes, because many of these enzymes have relaxed substrate specificity and process genetically encoded substrates.3 Among the four different types of phylogenetically divergent lanthipeptide synthetases that have been discovered,4-9 the class II lanthipeptide synthetases (LanM enzymes) have been the most thoroughly studied. These enzymes employ an N-terminal dehydratase domain (pfam ID PF13575) to catalyze the ATP-dependent dehydration of Ser/Thr residues via a phosphorylated intermediate, and a C-terminal Zn-dependent cyclase domain (PF05147) to catalyze the Michael-type addition leading to the thioether rings.10-13 Interestingly, extensive mechanistic and kinetic studies of LanM enzymes performed in recent years have shown that these enzymes exhibit drastic differences in their kinetic properties, substrate specificities, and directionalities.14-18

Recent comparisons between the

haloduracin  synthetase (HalM2) and the prochlorosin synthetase (ProcM) illustrate just how different the functional properties of LanM enzymes can be. HalM2 installs modifications into its substrate peptide, HalA2, in an N- to C-terminal direction with high catalytic efficiency and substrate specificity.14, 16, 19

In contrast, ProcM accepts 30 different precursor peptides as substrates, suffers a drastically

reduced catalytic efficiency, and exerts less stringent control over the modification directionality.2, 15-17 Clearly, different sets of mechanistic and biophysical factors seem to be important in defining the function of these two class II lanthipeptide synthetases. ACS Paragon Plus Environment


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What are the mechanistic and biophysical factors that contribute to the functional heterogeneity observed in LanMs? In addition to the primary amino acid sequence of the LanA precursor9, 20 and the coordinating ligands to the Zn2+ ion in the cyclase domain,17 which can dictate the preferred thioether cyclization topology, the LanM/LanA binding mode has been suggested to contribute to functional heterogeneity.14, 16, 18 Most notably, the observation that post-translational modification directionality, catalytic efficiency, and cyclization fidelity change in some systems when the leader peptide is mutated or physically removed from the core peptide, provides strong evidence that intermolecular LanM/LanA interactions and conformational changes within the LanM/LanA Michaelis complex play an important role in the observed functional properties and heterogeneity of these systems.18, 21-22 Moreover, recent kinetic studies have provided strong evidence that biophysical docking interactions between the core peptide of the LanM-bound LanA peptide gate the net rates of the chemical transformations measured in the steady state, and that these net rates change as a function of the structure of the maturing LanA intermediate.16 The simplest explanation to account for these findings is that the structure of the maturing intermediate influences the binding affinity of the intermediate to the LanM active sites. This, in combination with the spatial orientation of the core peptide relative to the two LanM active sites (which is presumably established by the leader peptide binding mode), serves to establish the preferred sequence of chemical events in the multistep maturation process. While the involvement of sets of specific intermolecular interactions between LanM and LanA is an attractive hypothesis to explain the functional heterogeneity observed within the LanM enzyme family, experimental validation of this hypothesis has proven difficult. To date, the cytolysin synthetase (CylM) from Enterococcus faecalis is the only LanM for which a high resolution crystal structure has been solved (Figure 1B).23 The enzyme has a two-lobed domain architecture where the active sites of the N-terminal dehydratase domain and C-terminal cyclase domain are separated by approximately 40 Å.

The large distance between the two active sites necessitates a biophysical model involving

extensive conformational sampling of the LanM/LanA complex, or entirely different LanA binding modes to the two functional domains. Although no co-crystal structure of CylM with a bound precursor peptide is available, an antiparallel 3-stranded -sheet located in the cyclase domain near the interface with the dehydratase domain has been suggested to contribute to precursor peptide binding (Figure 1B).23 This structural element (formed by CylM residues Ile666  Leu690) is reminiscent of the winged helix-turnhelix motif (termed the RiPP recognition element, RRE)24 that has been found recently in a number of different RiPP biosynthetic enzymes.25-29 Interestingly, biochemical studies of LanM enzymes that have been split into separate dehydratase and cyclase domains have shown that some LanM cyclases are competent enzymes when presented with dehydrated precursor peptides,17, 30 while others are not.31 Thus, while the β-stranded region of the cyclase domain may indeed be an important (albeit untested) ACS Paragon Plus Environment

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structural element in LanA binding, leader peptide binding and efficient dehydration and cyclization are likely to involve additional intermolecular interactions between LanM and LanA. The dearth of structural studies on lanthipeptide synthetases (and other RiPP biosynthetic enzymes) in the presence of their precursor peptide substrates is an impediment to the full realization of the potential of these enzymes as catalysts for making structurally complex peptides from genetically encoded materials. Clearly, a full understanding of the intrinsic structural dynamics of lanthipeptide synthetases and how this relates to the mechanisms that provide specificity and catalytic efficiency, should help to assess the usefulness of these enzymes for designed biocatalytic processes. In order to address these needs, we describe here the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate structural dynamic properties and intramolecular interactions in the model class II lanthipeptide synthetase, HalM2 (Figure 1C). The HDX-MS approach has the benefit of measuring the structural dynamic properties of LanM and LanM/LanA protein-peptide interactions using small amounts of unlabeled molecules under near native conditions in buffered aqueous solvents, which are expected to report faithfully on physiologically relevant biophysical interactions. HDX-MS has been used previously to investigate leader peptide binding to the kinase domain of class IV lanthipeptide synthetases32 and to the B1 scaffolding protein involved in lasso peptide biosynthesis.33 In this work, we greatly expand upon the HDX-MS approach to identify structural elements in HalM2 that are critical for efficient substrate binding and catalysis, to reveal allosteric communication between the two active sites of the enzyme in response to ligand binding, and to detect perturbations to enzyme dynamics mediated by the HalA2 core peptide. Aided by multiple amino acid sequence alignments, secondary structure predictions, homology modeling and the high-resolution structure of CylM, we exploit these HDX data for the construction of a panel of HalM2 variant enzymes that exhibit a range of distinct perturbations to normal HalM2 catalysis and that illustrate the malleable nature of these multifunctional enzymes. Cumulatively, the data paint a portrait of a conformationally dynamic enzyme that undergoes a variety of structural transitions in response to ligand binding. These data exemplify the vast potential of the HDX-MS approach for identifying mechanistically relevant conformational changes in RiPP biosynthetic enzymes – even in the absence of a high-resolution structure. As such, the HDX-MS approach is expected to find wide utility in the characterization of many other structurally dynamic RiPP biosynthetic enzymes. Results and Discussion Brief Overview of HDX-MS Approach: In this work, we employed a continuous exchange, bottom-up HDX-MS analysis34-36 in order to detect perturbations to HalM2 structural dynamics under different ligand-bound conditions, and to localize those perturbations to specific regions of the HalM2 enzyme. ACS Paragon Plus Environment


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In this workflow, undeuterated enzyme is diluted into a reaction mixture containing high D2O content, and the reaction is periodically sampled to assess the time-dependence of hydrogen-deuterium exchange (HDX). In this continuous exchange experiment, the protein backbone amide HDX rate is strongly influenced by the local hydrogen bonding, solvent accessibility, and neighboring amino acid residues.37-39

Proteins undergo dynamic conformational changes, where transient disruption of

hydrogen bonding networks exposes the backbone amides to deuterium exchange – leading to a timedependent change in the amount of deuterium uptake as the protein samples partially unfolded conformations. As such, perturbations (e.g. upon ligand binding) to the amide HDX rate measured in continuous exchange experiments typically reflect perturbations to the structural dynamics of the secondary and tertiary structure of the protein. Prior to MS analysis, the sample is proteolytically digested (bottom-up) such that the deuterium exchange can be measured independently into peptides derived from different regions of the enzyme.35 Thus, this approach effectively allows enzyme structural dynamics to be mapped and measured on local spatial scales. HalM2 is a structurally dynamic enzyme: Given that very little is currently known about the global conformational dynamic properties of any LanM enzyme, our initial experiments focused on the free HalM2 enzyme under assay conditions that support high-level HalM2 activity.16 Accordingly, bottomup, continuous exchange HDX studies were conducted using established procedures to maximize the deuterium content of the mass spectrometry-detected peptides.34, 40 The HDX-MS workflow resulted in the reproducible, high confidence detection of 235 HalM2-derived peptic peptides spanning over 90% of the full length HalM2 sequence (Figure 2A). A detailed description of the overall reproducibility of the assay as well as relevant chromatographic, mass spectrometric, and HDX data for every peptide included in the analysis is provided in the Supporting Information. The time-dependent fractional uptake of deuterium (Figure 2B) clearly shows that HalM2 contains many conformationally dynamic regions that are susceptible to rapid deuterium exchange within the first 30 s of the exchange reaction. Interestingly, many of the most dynamic portions of HalM2 are located on the active face of the molecule between the active sites of the dehydratase and cyclase domains (Figure 2C), suggesting that these regions could be involved in HalA2 binding and/or the formation of enzyme conformations that promote efficient post-translational modification. Of particular note is a large loop (disordered in the CylM structure) spanning HalM2 residues Pro349 – Pro405 in the N-terminal dehydratase domain. This large loop of approximately 50 amino acid residues (herein dubbed the Pro349-Pro405 loop) is conserved in the LanM family and also contains a moderately conserved Thr/Ser-Asp dipeptide motif flanked by predicted -stranded elements (Figures S3). Additional disordered loop regions of high deuterium uptake in the dehydratase domain include residues Trp491-Met497 located in the kinase activation (KA) domain23 which interacts with the ACS Paragon Plus Environment

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kinase activation loop (Leu314-Phe329), as well as residues His110-Phe130 located immediately Cterminal to the k2 helix (using lipid kinase nomenclature).41 Both the KA domain and the His110Phe130 region are unique to LanMs among the enzymes that share the lipid kinase fold.23 In addition, significant deuterium uptake was observed in the putative precursor peptide binding -sheet of the cyclase domain (Ile627-Glu635),23 as well as in loops Phe681-Ala693, Leu777-Leu786, and Val835Phe846 of the cyclase domain, which appear to frame an entry point to the active site Zn2+ ion (Figure S4). The Leu777-Leu786 loop is directly adjacent the catalytic His791 residue that protonates the enolate formed by Michael-type addition during the thioether ring forming reaction (Figure 1A).12 The Val835-Phe846 loop is directly adjacent to the Cys848 residue that serves as one of the three ligands to the catalytic Zn2+ in the cyclase active site.12 Functional assignments have not yet been proposed for most of these highly dynamic loops in HalM2. These results demonstrate that HalM2 is indeed a highly dynamic enzyme, and they highlight the power of HDX-MS to rapidly reveal potentially important, previously overlooked functional regions of dynamic enzymes under near native conditions – even when no high-resolution structure is available. Ligand binding induces HDX changes in HalM2 that guide functional analysis: Intrigued by the initial HDX studies of free HalM2, which seemed to indicate the presence of structurally dynamic elements in spatial locations that were potentially critical for HalM2 function, we next investigated the structural perturbations to HalM2 in the presence of adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP, a nonhydrolyzable ATP analogue), ATP, the full length HalA2 precursor peptide, and the HalA2 leader peptide (HalA2-LP, comprised of HalA2 residues Met1-Gly36, Figure 1). The simple hypothesis for these studies is that nucleotide and/or peptide binding will induce local and/or long-range perturbations in the structural dynamics (and hence, in the amide HDX rate) of the HalM2 backbone that might reflect intermolecular binding interactions and intramolecular conformational changes. Peptides exhibiting a significant exchange difference between states were determined as described previously42 by summing the exchange difference for each peptide at each exchange time point. As detailed in the Supporting Information, we estimated an average uncertainty of 0.087 Da in our uptake difference measurements between replicate samples, in good agreement with similar estimates made by others42-43 and highlighting the sensitivity of the approach for detecting even slight changes in protein conformational dynamics. The differentially exchanging peptides were then mapped onto the HalM2 homology model with the assistance of the Deuteros software package recently developed by Politis et. al (Figure 3).44 AMP-PNP binding reveals long-range structural communication: We first examined the effects of AMPPNP binding on the HDX properties of HalM2 (Figure 3A-B). Activity assays confirmed that AMP-PNP ACS Paragon Plus Environment


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is an effective inhibitor of HalM2 (Figure S5). Upon AMP-PNP binding, decreases in deuterium uptake were observed in the N lobe of the dehydratase domain near the nucleotide binding site in peptides spanning Ile235-Ala251, as well as in the P loop (Gly198-Gln205), which harbors residues involved in phosphate binding (Asp199 and His201). Weaker, though consistently enhanced protection was also observed in peptides covering secondary structural elements immediately adjacent to the P loop as well as in all 15 peptides covering the putative N-terminal helix of the capping domain (Trp24-Glu39). Thus, these portions of HalM2 also seem to become slightly more organized upon AMP-PNP binding, though our assay would require additional sensitivity for a conclusive demonstration. In the kinase activation (KA) subdomain, which is unique to LanM enzymes (Figure 2D), peptide Ile454-Tyr464 containing the catalytic Arg455 and Thr461 residues involved in phosphate elimination (Figure 1), becomes significantly more dynamic upon AMP-PNP binding. Thus, the HDX studies reveal subtle conformational fluctuations within the dehydratase active site that could be relevant to catalysis. In other less functionally-defined regions of the dehydratase domain, peptides within the Pro349-Pro405 loop of the C lobe (Gly344-Leu357, Ile360-Thr378, Val365-Thr378) become more protected from HDX, and peptides spanning Ala572-Ser592 that demarcate the beginning of the cyclase domain become more susceptible to HDX. Quite surprisingly, the most significant and obvious region of HDX perturbation upon AMP-PNP binding to the dehydratase domain occurred within the HalM2 cyclase domain. The strong perturbation localized to residues Glu635-Gly652, which encompass the entire third strand of the antiparallel -sheet in the cyclase domain of the HalM2 homology model, as well as the loop that connects this strand to the downstream -helix (Figure 3 A-B, and Figure 4A). The coverage of this region by multiple HalM2derived peptides (see Figure 2A), all of which exhibit the same trend in decreased deuterium uptake in the presence of AMP-PNP, strongly support a considerable structural organization of this region upon AMP-PNP binding. An additional loop spanning residues Asn940-Leu950, which interacts with the sheet in the HalM2 homology model (Figure 4A), also becomes significantly protected upon AMP-PNP binding. As examined further below, the -sheet within the cyclase domain appears to interact directly with the HalA2 peptide in a way that facilitates efficient post translational modification by HalM2. Thus, the rigidification of this secondary structural element upon AMP-PNP binding site may prime the enzyme for productive interactions with its precursor peptide, and clearly suggests the potential for long-range structural communication across the active face of the enzyme mediated by ligand binding. ATP hydrolysis in the dehydratase domain triggers a global structural relaxation: In the presence of 5 mM ATP (Figure 3C-D), the two most significant regions of protection from HDX were again localized to the N lobe of the dehydratase domain (Ile235-Ala251) and to the -sheet of the cyclase domain ACS Paragon Plus Environment

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(Glu635-Gly652). The protection against HDX within the -sheet was even more pronounced in the [HalM2:ATP] state, with almost no detectable exchange occurring over the 4 h time course of the HDX assay (Figure 4B). Additionally, organization of the -sheet in the [HalM2:ATP] state rigidifies an adjacent loop element (Ala693-Phe701) that interacts directly with the -sheet in the HalM2 homology model (Figure 4A). Thus, both AMP-PNP and ATP binding to the dehydratase domain result in a significant organization of structural elements within the cyclase domain, near the putative peptide binding -sheet. The most characteristic feature of the [HalM2:ATP] state, however, is the significantly enhanced deuterium uptake scattered throughout both functional domains of the enzyme (Figure 3C-D). In the dehydratase domain, these include peptides within the N-terminal helical domain (His110-Phe144), the P loop and neighboring elements in the N lobe (Thr191-Gln230), as well as almost every peptide within the Pro349-Pro405 loop, the kinase activation domain, and the k10-k11 linker. In the cyclase domain, relaxation occurs in peptides containing the active site Zn2+ ligands (Asn875-Leu900), within helical elements that pack against the -sheet (Phe657-Ala693) and on the back side of the cyclase domain (Tyr906-His931), as well as within dynamic loop regions near the Zn2+ ion (Val835-Phe846, Leu777-Leu786, and Thr932-Leu939). To test whether the unexpectedly dramatic enhancement in deuterium uptake across HalM2 was the result of ATP hydrolysis, we constructed a triple mutant enzyme

(HalM2-K221M/E315Q/R455M)

lacking

catalytic

residues

that

contribute

to

the

phosphoryltransfer and (Lys221 and Glu315) and elimination (Arg455) reactions of peptide dehydration.13 Activity assays confirmed the lack of peptide dehydration and ATPase activity in the triple mutant (Figure S6). Moreover, when the HDX studies were repeated for this mutant in the presence and absence of 5 mM ATP, deuterium uptake was no longer enhanced in the presence of ATP (Figures 5 and S6), suggesting that the increased uptake observed in the wild type enzyme is likely the result of ATP hydrolysis. From the estimated ATP hydrolysis rate (0.13 min-1, Figure S6B), each wt HalM2 molecule would be expected to hydrolyze approximately 4 ATP molecules over the 30 min course of the exchange reaction shown in Figure 5. Cumulatively, these data suggest that ATP hydrolysis triggers perturbations in the secondary and tertiary structure of HalM2. While previous studies have shown that LanM enzymes are capable of processing phosphorylated peptides in the absence of ATP,11,

16

the involvement of ATP-driven conformational changes in promoting efficient

catalysis has never been thoroughly investigated. HalA2 binding also triggers global structural perturbations in HalM2: Previous biochemical studies have provided support for a conformational selection mechanism for LanMs, in which leader peptide binding shifts the population of LanM conformations towards a state with enhanced catalytic activity.21, 45 In an ACS Paragon Plus Environment


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attempt to identify and probe these potentially unique conformations, we conducted HDX reactions in the presence of AMP-PNP and the full length HalA2 precursor peptide (Figure 3E-F) in order to assess the effects of HalA2 binding on the structural dynamic properties of the [HalM2:AMP-PNP] complex. Using previous estimates for the HalA2 dissociation constant (𝑘𝑜𝑓𝑓 = 18 min-1)16 and the binding affinity reported in Table 1 (𝐾𝑑 = 0.7 M), the 𝑘𝑜𝑛 for HalA2 can be estimated as 26 M-1min-1. Thus, while the precursor peptide will dissociate from the enzyme many times over the course of the 240 min HDX assay, the binding equilibrium should favor the fully complexed [HalM2:AMP-PNP:HalA2] state by a factor of approximately 22 fold at the 15 M HalA2 concentration used in the assay. Interestingly, similar to what was observed with the free enzyme in the presence of ATP, HalA2 binding to [HalM2:AMPPNP] triggers an overall structural relaxation of the complex (compare Figures 3D and 3F), lending strong support to a biophysical model where peptide binding modulates the conformational dynamic properties of the enzyme – perhaps poising the system to sample catalytically productive conformations. The most pronounced relaxation in the dehydratase domain occurs within the Nterminal helical capping domain, the N lobe elements comprising the nucleotide binding site, the Pro349-Pro405 loop of the dehydratase domain, and the k10-k11 helices, which form part of the interface with the cyclase domain. Enhanced deuterium uptake is likewise widespread in the cyclase domain, but is most prominent in the -sheet and in elements surrounding the active site Zn (Leu777Leu786, Val835-Phe846 and Ile927-Leu939). Thus, while AMP-PNP and ATP binding rigidify and organize the -sheet, HalA2 precursor peptide binding re-introduces some flexibility into this element (Figure 4B), though even in the fully complexed [HalM2:AMP-PNP:HalA2] state, the sheet remains quite protected from deuterium exchange relative to the free enzyme. It is tempting to speculate that the variable structural dynamics of the -sheet are somehow relevant to thioether cyclization catalysis by HalM2, which must be capable of accommodating the different partially cyclized core peptide structures that form during precursor peptide maturation. While a global structural relaxation is the most defining feature of HalA2 binding, several conspicuous regions of HalM2 underwent prominent decreases in deuterium uptake. These include the His110-Phe130 loop (unique to LanMs among proteins with the lipid kinase fold)23 in the N-terminal helical capping domain, an -helical region (Ala335-Phe343) that separates the activation loop from the Pro349-Pro405 loop, and peptide Met389-Glu399 within the Pro349-Pro405 loop that is predicted to have -strand character (Figure S3). Biochemical studies reported below demonstrate that each of these motifs plays a critical role in either HalA2 binding or HalM2 catalytic function. In addition, noticeably enhanced protection was also observed in the kinase activation domain (residues Gln453Phe467) and in the kinase activation loop (Tyr290-Leu297), though the HDX difference measured for these elements fell just below the stringent criteria for significance in our analysis. These elements ACS Paragon Plus Environment

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contain several catalytic residues involved in the phosphorylation (Asp396) and phosphate elimination (Arg455 and Thr461) reactions catalyzed by the dehydratase domain.13, 23 Finally, we also investigated HDX perturbations upon binding of HalA2 to HalM2 in the absence of AMP-PNP (Figure S17). These experiments yielded similar results as the comparison shown in Figure 3E-F, providing additional support that HalA2 binding triggers global structural changes throughout the enzyme. The HalA2 core peptide perturbs HalM2 structural dynamics: HDX studies performed in the presence of the HalA2 leader peptide (HalA2 residues Met1-Gly36) confirmed that most of the structural perturbations observed in the [HalM2:AMP-PNP] complex upon full length HalA2 binding are attributable to interactions between HalM2 and the leader peptide. This is evidenced by the minimal HDX differences across most of the protein sequence when the [HalM2:AMP-PNP:HalA2-LP] state is compared with the [HalM2:AMP-PNP:HalA2] state (Figure 3G-H). Thus, the HDX data is consistent with previous studies on HalM2 that have shown that bimolecular binding interactions with HalA2 are governed mainly by the leader peptide.45 Nevertheless, slight differences between the structural dynamics of the HalA2- and HalA2-LP-bound states are observable. For peptides Ile235-Ala251 (N lobe), Ala335-Phe343 (C lobe), Ile360-Thr378 (Pro349-Pro405 loop), and Glu635-Gly652 (cyclase sheet), the full length HalA2 exhibits a stronger perturbation than the HalA2 leader peptide. Moreover, additional regions of strong HDX protection in the presence of full length HalA2 map to the -sheet (peptide Ile627-Met633) and to peptides spanning residues Asn875-Leu900 that harbor two of the active site Zn2+ ligands (Figure 4A). These HDX differences between the HalA2- and HalA2-LP bound states could be mediated either by direct interactions with the HalA2 core peptide and/or by propagation of perturbations induced by core peptide interactions with more distal regions of the protein. Regardless of the exact nature of these interactions, the HalA2 core peptide clearly influences the structural dynamics of the fully complexed enzyme, providing evidence to support previous studies that LanM enzymes are capable of recognizing both leader and core peptide regions of their precursor peptides.18, 21-22, 45

Biochemical studies validate functional relevance of HDX hot spots: The HDX-MS analyses discussed above revealed a number of sequence elements in HalM2 that have not been previously implicated in LanM function. In an attempt to define their biochemical roles, we constructed a number of HalM2 variants by replacing some of these dynamic elements with short GlySer linkers (Figure 6). We focused our attention on the unique sequence insertion in the N-terminal capping domain (variants HalM2GS110-115 and HalM2-GS116-123) that becomes significantly organized upon HalA2 and HalA2-LP binding, regions in the C lobe within and surrounding the Pro349-Pro405 loop (HalM2-GS331-340, HalM2-GS341ACS Paragon Plus Environment


348,

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and HalM2-GS389-396), the putative interface between the kinase activation domain and the

activation loop (HalM2-GS491-497), and the -sheet in the cyclase domain (HalM2-GS635-644). Note that in our naming convention, the subscript refers to the consecutive HalM2 amino acid residues that were replaced with the repeating Gly-Ser dipeptide. Each variant protein was expressed and purified to near homogeneity in yields similar to the wild type HalM2, and their purities and molecular weights were confirmed by LC-ESI-MSI analysis (Figure S7). The variant enzymes were subsequently characterized by thermal denaturation assays (Table 1, Figure S8), fluorescence polarization binding studies (Table 1, Figure S9), and activity assays (Table 1, Figure 7 and S10-S16). A summary of these results and our mechanistic interpretation follows. Thermal denaturation studies revealed only slight perturbations in the Tm values of the variant enzymes but, overall, the measured values are consistent with well folded proteins, and there was no apparent correlation between the measured Tm values and either the catalytic activities or peptide binding affinities of the variant enzymes. The binding affinity of the fluorescein-labeled HalA2 leader peptide (fluoro-HalA2-LP) for the HalM2-GS635-644 variant (0.9 M) is very similar to wild type HalM2 (0.7

M), suggesting that the 3rd strand of the -sheet in the cyclase domain makes minimal

contributions to bimolecular substrate binding and may not act as a RiPP recognition element in the same manner as the winged helix-turn-helix described previously in other systems. This minimal contribution to HalA2 binding is supported by the similar HDX properties of peptide Glu635-Leu651 in the [HalM2:AMP-PNP] and [HalM2:AMP-PNP:HalA2-LP] states (Figure 6B), which illustrate that the HalA2 leader peptide does not trigger any additional organization within this element. Interestingly, upon binding to full length HalA2, the adjacent -strand of the sheet (peptide Ile627-Met633) becomes organized relative to the [HalM2:AMP-PNP:HalA2-LP] state (Figure 4A), suggesting that a direct interaction between the HalA2 core peptide and the cyclase domain -sheet is possible. In line with this hypothesis, the HalM2-GS635-644 enzyme suffered from significantly reduced post-translational modification rates that can be attributed mainly to a reduction in cyclization rates (Figures 7 and S10, Table 1). Thus, while the -sheet in the cyclase domain may not contribute much to bimolecular HalA2 peptide binding, it could potentially engage in transient interactions with the HalA2 peptide within the Michaelis complex in order to promote efficient docking interactions and cyclization.16 The increased dynamics of the sheet upon HalA2 binding may provide a mechanism for the enzyme to accommodate the maturing structure of the core peptide during biosynthesis. Replacement mutations to the -helical region in the C lobe immediately adjacent to the Pro349Pro405 loop (the HalM2-GS331-340 and HalM2-GS341-348 variants) also did not significantly perturb fluoroHalA2-LP binding. Thus, despite the strong HDX protection in this region upon HalA2 and HalA2-LP binding (Figure 3), these regions do not appear to bind the HalA2 peptide directly. However, these ACS Paragon Plus Environment

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replacement mutations resulted in severe disruptions of wt HalM2 activity (Figure 7). The HalM2-GS341348

variant consumed almost no HalA2 substrate over the 8 h time course of the assay (see Figure S11

for extended time course), whereas the HalM2-GS331-340 enzyme produced only minimal quantities of partially modified intermediates (Figure S12). Our current model is that the rigidification of these helices upon HalA2 binding may play some integral role in propagating intramolecular conformational changes that are necessary for efficient catalysis to proceed. Indeed, the Phe331-Leu338 helix interacts directly and extensively with the -sheet in the cyclase domain and is immediately downstream of the activation loop that harbors several critical catalytic residues required for the dehydration reaction. The highly conserved Ser341-Leu348 region (see Figure S3) is directly adjacent to catalytic residues (Asp296 and His298) in the dehydratase active and also defines the start of the Pro349-Pro405 loop, whose dynamic properties are highly sensitive to ligand binding. Studies are currently underway to more thoroughly delineate the precise biochemical structural dynamics and function of these elements. Mutations within the Pro349-Pro405 loop (HalM2-GS389-396) and in the kinase activation domain (HalM2-GS491-497) led to moderate decreases in fluoro-HalA2-LP affinity (to 4.2 M and 3.8 M, respectively). This observation is consistent with the involvement of both elements in defining a wellordered HalA2 binding site – perhaps involving the Pro349-Pro405 loop, and is supported by the increased HDX protection in these regions upon HalA2 binding (Figure 3E-F, Figure 6B). Interestingly, the catalytic activity of both of these variants is perturbed in a manner that results in the formation of phosphorylated intermediates (Figure 7). In wt HalM2, the phosphoryl transfer reaction is strongly coupled to the phosphate elimination step within the dehydratase active site, such that the Ser/Thr residues are dehydrated without release of a phosphopeptide intermediate (Figure 1).16 The two reactions of the net dehydration sequence become uncoupled in the HalM2-GS389-396 and HalM2-GS491497

mutants, perhaps because of perturbations to enzyme conformations that stabilize core peptide

docking in the dehydratase active site. The HalM2 homology model predicts the presence of a short

-strand in the Trp491-Met497 loop that interacts with the kinase activation loop. When this interaction is disrupted, the activation loop may lose the ability to effectively position catalytic residues in the activation loop upon core peptide docking. Similarly, PSIPRED predicts -stranded character in HalM2GS389-396 (Figures S1 and S3), suggesting that this portion of the loop may either help to bind HalA2 directly and/or nucleate some sort of tertiary structure that facilitates efficient dehydration catalysis. Finally, among the mutants tested in the work, the HalM2-GS110-115 and HalM2-GS116-123 variants (located in the N-terminal capping domain of the dehydratase) exhibited the most drastic reductions in fluoro-HalA2-LP binding affinity. As noted above and in the CylM structural studies,23 this portion of HalM2 lies in a disordered sequence region that is unique to LanMs. For HalM2-GS116-123, the Kd for HalA2 increased approximately 15 fold to 11 M. In terms of catalytic activity, however, the HalM2ACS Paragon Plus Environment


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GS116-123 variant was not that dissimilar from wt HalM2 under our assay conditions (containing 1 M enzyme and 50 M HalA2), yielding complete conversion to fully modified HalA2 after 2 h (Figure 7) and a similar product distribution as the wild type enzyme after 10 min (Figure S15). Thus, this replacement mutation seems to solely affect bimolecular interactions with HalA2, and does not appear to modulate any enzyme conformations that influence catalysis – an intriguing possibility for future LanM engineering applications. In contrast, the binding affinity of fluoro-HalA2-LP for the HalM2-GS110115

enzyme could not be measured with our assay, and this enzyme exhibited only minimal activity after

8 h, even when the HalA2 peptide was increased to its solubility limit (~ 150 M under our assay conditions, Figure S16). Thus, it is very likely that this portion of the N-terminal helical capping domain of HalM2, which is unique to LanM enzymes, binds directly to the HalA2 leader peptide or is absolutely essential for the formation of a HalM2 conformation that enables precursor peptide binding. Conclusions:

Many RiPP biosynthetic enzymes, such as the class II lanthipeptide synthetases

investigated in this study, are multifunctional enzymes with relaxed substrate specificity that install sets of post-translational modifications into precursor peptide substrates.

The mechanisms by which

biosynthetic fidelity (and sometimes, biosynthetic flexibility) is achieved in these enzyme systems is an interesting and challenging area of research that has implications in bioengineering and synthetic biology applications. The potential utility of lanthipeptide synthetases for generating new-to-nature cyclic peptides with novel functions has been demonstrated in several recent and elegant molecular engineering applications.46-49 Among other mechanisms that have been demonstrated to contribute to substrate specificity, biosynthetic fidelity, and catalytic efficiency in these systems, it has been proposed that a biophysical model involving extensive conformational sampling of the LanM:LanA Michaelis complex can be used to rationalize much of the existing biochemical data.16 Here, we probe this hypothesis using hydrogen-deuterium exchange mass spectrometry and provide the first direct evidence that HalM2 is indeed a conformationally dynamic molecule that undergoes structural dynamic changes in the presence of its ligands. Our data reveal significant perturbations in the equilibrium distribution of HalM2 conformations in the presence of nucleotide ligands (AMP-PNP and ATP), the full length HalA2 precursor peptide, and the HalA2 leader peptide. These changes include dramatic increases in the structural dynamics across the active face of the enzyme in the presence of ATP or peptide, which may poise the enzyme for precursor peptide binding and enable access to catalytically competent conformations, respectively.

Moreover, nucleotide

binding to the dehydratase active site triggers a pronounced organization of the three stranded antiparallel -sheet in the cyclase domain, providing clear evidence for allosteric communication between the dehydratase and cyclase domains. Comparison of the HalA2 and HalA2-LP bound states ACS Paragon Plus Environment

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also provides clear evidence that the HalA2 core peptide influences the structural dynamics of the enzyme – especially within the -sheet and the active site of the cyclase domain. Thus, HDX-MS may prove to be an ideal technique to examine the mechanism of core peptide recognition by LanM enzymes, which has remained elusive. In addition, we provide the first evidence that HalM2 employs a unique sequence element in the N-terminal -helical domain in order to bind to HalA2. Thus, the mode of precursor peptide recognition by LanM enzymes seems to be fundamentally different from class I lanthipeptide synthetases and many other RiPP biosynthetic enzymes, which employ the winged helix-turn-helix RiPP recognition element.24-29

Finally, we show that mutations to many of the

structurally dynamic elements in HalM2 (which have not previously been implicated in LanM function) result in enzymes that exhibit a range of catalytic perturbations. Thus, this work illustrates the power of the HDX-MS approach for revealing previously overlooked structural elements in lanthipeptide synthetases that are functionally relevant. The approach should find wide utility in the characterization of other RiPP biosynthetic enzymes, which operate along similar mechanistic paradigms to the class II lanthipeptide synthetases.3 Acknowledgement. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Fonds de Recherche du Québec Nature et Technologie, the Canadian Foundation for Innovation, and McGill University start-up funds. Y.H. and K.A.U. contributed equally to this work. Supporting Materials Available. Detailed methods for all biochemical assays, mass spectrometry analyses, molecular genetics and bioinformatics approaches.

Supplemental figures, sequence

alignments, and mass spectrometry data. These are available free of charge.



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Table 1. Summary of biochemical data for wild type HalM2 and its variant enzymes constructed in this work. Enzyme

𝑻𝒎 (ºC)

𝑲𝒅 (M)

𝑭𝒄𝒚𝒄

𝑭𝒅𝒆𝒉𝒚𝒅

𝑭𝒕𝒐𝒕𝒂𝒍

wt HalM2

56.2 ± 0.06

0.7 ± 0.2

3.81 ± 0.02

6.70 ± 0.01

0.96

HalM2-GS116-123

52.5 ± 0.01

11 ± 5

3.68 ± 0.02

6.51 ± 0.03

0.93

HalM2-GS491-497

52.8 ± 0.01

3.8 ± 2.2

2.53 ± 0.2

5.9 ± 0.01

0.77

HalM2-GS635-644

52.5 ± 0.05

0.9 ± 0.3

2.40 ± 0.13

5.5 ± 0.12

0.72

HalM2-GS389-396

51.6 ± 0.02

4.3 ± 0.9

1.64 ± 0.02

4.39 ± 0.04

0.55

HalM2-GS110-115

52.1 ± 0.01

𝑁.𝐷.

0.24 ± 0.07

0.9 ± 0.1

0.10

HalM2-GS331-340

53.2 ± 0.02

2.01 ± 0.8

0.22 ± 0.003

0.9 ± 0.05

0.10

HalM2-GS341-348

53.6 ± 0.3

1.0 ± 0.35

0.14 ± 0.006

0.58 ± 0.01

0.07

The extent of cyclization (𝐹𝑐𝑦𝑐), extent of dehydration (𝐹𝑑𝑒ℎ𝑦𝑑), and total extent of reaction (𝐹𝑡𝑜𝑡𝑎𝑙), were calculated as described in the Supporting Information from mass spectra taken after 2 h (Figure 7). 𝐹𝑐𝑦𝑐 has a maximum value of 4 (one for each thioether ring in the fully modified HalA2 product). 𝐹𝑑𝑒ℎ𝑦𝑑 has a maximum value of 7 (one for each dehydration site in the fully modified HalA2 product). 𝐹𝑡𝑜𝑡𝑎𝑙 is the sum of 𝐹𝑐𝑦𝑐 and 𝐹𝑑𝑒ℎ𝑦𝑑 divided by 11 and is intended to quantify the extent of reaction on a normalized scale for all HalM2 variants. The thermal denaturation data for determining 𝑇𝑚 and equilibrium binding data for determining 𝐾𝑑 are shown in Figures S8 and S9, respectively.


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Figure 1. Overview of Class II Lanthipeptide Biosynthesis. A) Chemical mechanism of class II lanthipeptide biosynthesis. Ser = serine, Thr = threonine, Dha = 2,3-didehydroalanine, Dhb = (Z)-2,3didehydrobutyine, Lan = lanthionine, MeLan = methyllanthionine. B) X-ray crystal structure of CylM with AMP (PDB entry 5DZT, shown in stick format) bound to the dehydratase domain and Zn2+ (green sphere) bound to the cyclase domain. The putative LanA-binding -sheet is indicated. C) Posttranslational maturation of the HalA2 precursor peptide catalyzed by HalM2. The Ser/Thr residues highlighted in yellow are dehydrated by HalM2 and the Cys residues involved in thioether ring formation are highlighted in purple. The cyclization topology of the fully modified HalA2 precursor peptide is shown.



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Figure 2. Solvent Deuterium Exchange Properties of HalM2. A) HalM2 coverage map, where each bar represents a HalM2-derived peptide colored according to its relative fraction deuterium uptake. These 235 peptides were reproducibly detected in each of the ligand bound states investigated in this study, allowing direct comparison of localized HDX differences across multiple samples. The relevant measurables for each peptide (theoretical and measured m/z, detected charge states, retention time, and deuterium uptake) in each state are provided as supporting materials. B) Fractional deuterium uptake as a function of both HalM2 amino acid residue number and exchange time. Many of the HalM2 backbone amides are located within highly dynamic regions of the enzyme, and exchange quickly within ACS Paragon Plus Environment

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the first 30 s of the exchange reaction. The data from the 5 min exchange time point are plotted in panel A. C) Conformationally dynamic regions of HalM2 are mapped onto the HalM2 homology model. See the Supporting Information for details on homology model construction. The dynamic regions map mostly to loops that connect the more structurally conserved LanM secondary structural elements (Figures S1-S3).23 Most of the His110-Phe130, Pro349-Pro405, Leu778-Leu786, and Phe681-Ala693 loops were not ordered in the CylM crystal structure and, thus, their positions in the HalM2 homology model should be considered approximate. D) The standard domain nomenclature for LanM enzymes used throughout this study.23 The domains are also mapped onto the primary sequence in panel A.



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Figure 3. Deuterium uptake difference maps reveal perturbations to HalM2 structural dynamics in the presence of ligands. Deuterium uptake differences were calculated in order to compare biochemical states [HalM2] with [HalM2:AMP-PNP] (panels A-B), [HalM2] with [HalM2:ATP] (panels C-D), [HalM2:AMP-PNP] with [HalM2:AMP-PNP:HalA2] (panels E-F), and [HalM2:AMP-PNP:HalA2-LP] with ACS Paragon Plus Environment

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[HalM2:AMP-PNP:HalA2] (panels G-H). The uptake differences were calculated as the sum of the differences for each peptide at each exchange time point (gray bars in panels A, C, E, and G)42 and were subsequently mapped onto the HalM2 homology model using Deuteros (B, D, F, and H).44 The 99% confidence intervals for significant exchange differences (dashed red lines in panels A, C, E, and G) were also calculated in Deuteros. In all panels, blue and red indicate peptides that undergo more and less deuterium uptake, respectively, as defined by the difference being calculated (shown at the top of panels A, C, E, and G). In all structures, AMP and Zn2+ are shown in space-filling format in green and gold, respectively.



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Figure 4. The dynamic HDX properties of the HalM2 cyclase domain. Panel A) HDX differences between the indicated states are plotted onto ribbon models of the HalM2 cyclase domain. The dehydratase domain has been removed for clarity. The Zn2+ ion is shown as a gold sphere. Elements shaded in blue and red undergo more and less deuterium exchange, respectively, according to the difference being calculated (indicated at the top of each structure). The most significant perturbations cluster within the -sheet (Ile616-Pro641), peptides harboring Zn2+ ligands (Asn875-Leu900), and various loop regions framing the entry point to the active site Zn2+ ion. The deuterium uptake plots for a portion of the -sheet (peptide Glu635-Leu651) are shown in panel B, where the differences in uptake between the various HalM2 biochemical states is obvious.


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Figure 5. Mutations in the HalM2 dehydratase active site reduce HDX in the presence of ATP. The deuterium uptake of wild type HalM2 and the HalM2-K221M/E315Q/R455M triple mutant (HalM2m) enzyme in the presence of 5 mM ATP are compared. The HDX of the [HalM2m:ATP] state is subtracted from the [HalM2:ATP] state, showing that the wt enzyme uptakes more deuterium than the mutant enzyme over the 30 min exchange reaction. In the absence of ATP, the two enzymes exhibited similar exchange properties (Figure S6).



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Figure 6. Mutations to dynamic regions of HalM2. Panel A shows the positions of the loop replacement mutations made in this study, mapped onto the HalM2 homology model. Each indicated segment of HalM2 to be mutated was replaced with a poly-GlySer linker of equivalent length (see Supporting Information for details). Panel B shows deuterium uptake plots for peptides within the regions that were mutated in the various biochemical states investigated in this study. Each of these regions exhibited significant perturbations in the presence of different ligands, thus suggesting a potential role for these elements in HalM2 function.


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Figure 7. Comparison of HalM2 variant enzyme activities. All reactions were conducted under the standard assay conditions given in the Supporting Methods. Mass spectra derived from the 2 h reaction time point are shown for the HalA2 9+ ion family. The unmodified HalA2 starting material, fully modified product, and the extent of cyclization of the various reaction intermediates are indicated. Phosphorylated intermediates (indicated with asterisks) were only observed in the reaction spectra of the HalM2-GS389-396 and HalM2-GS491-497 variants. Extended mass spectral time courses for each enzyme are provided in Supporting Figures S10-S16.



Page 26 of 29

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Insights into the Dynamic Structural Properties of a Lanthipeptide

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