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Substrate interactions guide cyclase engineering and lasso peptide diversification

Nature Chemical Biology (2024)Cite this article

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Abstract

Lasso peptides are a diverse class of naturally occurring, highly stable molecules kinetically trapped in a distinctive [1]rotaxane conformation. How the ATP-dependent lasso cyclase constrains a relatively unstructured substrate peptide into a low entropy product has remained a mystery owing to poor enzyme stability and activity in vitro. In this study, we combined substrate tolerance data with structural predictions, bioinformatic analysis, molecular dynamics simulations and mutational scanning to construct a model for the three-dimensional orientation of the substrate peptide in the lasso cyclase active site. Predicted peptide cyclase molecular contacts were validated by rationally engineering multiple, phylogenetically diverse lasso cyclases to accept substrates rejected by the wild-type enzymes. Finally, we demonstrate the utility of lasso cyclase engineering by robustly producing previously inaccessible variants that tightly bind to integrin αvβ8, which is a primary activator of transforming growth factor β and, thus, an important anti-cancer target.

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Fig. 1: Overview of lasso peptide formation and loop tolerance.
Fig. 2: Substrate loop and lasso cyclase interactions.
Fig. 3: FusC R321 (helix 11) governs intolerance to basic loops.
Fig. 4: Validation of the interactions between helix 18 and core position one.

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Data availability

All data and methods from this study are available in this article, Extended Data Figures and Tables, Supplementary Information and Supplementary Data. The RODEO-mined dataset of lasso peptide substrate–lasso cyclase pairs is included as Source Data File 1. The asparagine synthetase crystal structure referenced is found in the Protein Data Bank under code 1CT9. National Center for Biotechnology Information accession identifiers for proteins examined in this study are listed in Supplementary Table 2 with their corresponding core peptide sequences. They are also reprinted and linked here with their name (if named): FusC: WP_104612995.1, NbsC: WP_089507158.1, SnoC AIA02568.1, XgaC: WP_006452335.1, CapC: ABC39497.1, BreC: EGF94507.1, CK31C: (IV) WP_012286030.1, PadeC: WP_006678396.1, McjC: WP_256498469.1, KleC: WP_023288231, UboC: WP_060255687.1, BurC: YP_004028968.1, AtxC: WP_013479714.1, NopC: WP_171983240.1, CsegC: YP_003593636.1, StlaC: QYB25760.1, CitC: WP_078843845.1 and HalC: WP_068689552.1, WP_167473437.1, WP_141996100.1, PWC97452.1, MCC6364916.1, WP_018083434.1, GDY30479.1, WP_035534278.1, WP_128790276.1 and OPC80860.1, TAJ26290.1. Lasso cyclase structures were predicted using AlphaFold version 2.1.2 on the High-Performance Biological Computing Biocluster Resource at the Institute for Genomic Biology at the University of Illinois Urbana-Champaign. MD simulation data are available for Figs. 2 and 3 and Extended Data Figs. 5 and 6 in the Dryad repository: https://doi.org/10.5061/dryad.fttdz092h. Other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The hmmexcise.py Python script and the Python notebooks used to analyze and create images from the MD simulations are available at https://github.com/ShuklaGroup/lasso_rotaxane.git.

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Acknowledgements

This work was supported, in part, by grants from the National Institutes of Health (R01GM123998 to D.A.M. and R35GM142745 and R21AI167693 to D.S.). S.E.B was supported by a National Science Foundation Graduate Research Fellowship (DGE 21-46756) and the University of Illinois Urbana-Champaign Illinois Distinguished Fellowship. We thank A. Kretsch and X. Mi for helpful discussions, J. Hegemann for providing the xanthomonin expression plasmid and S. Dommaraju for customizing the HVR_excise script.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Susanna E. Barrett, Thomas A. Pires, Ruoyang Wang, Diwakar Shukla & Douglas A. Mitchell

  2. Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Susanna E. Barrett, Thomas A. Pires, Ruoyang Wang & Douglas A. Mitchell

  3. Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Song Yin & Diwakar Shukla

  4. Lassogen, Inc., San Diego, CA, USA

    Peter Jordan, John K. Brunson, Jessica Gordon-Nunez, Gabriella Costa Machado da Cruz, Christopher Rosario, Bethany K. Okada, Kelsey Anderson & Mark J. Burk

  5. Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Diwakar Shukla

  6. Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Diwakar Shukla

  7. Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Douglas A. Mitchell

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Contributions

D.A.M. conceived the project. D.A.M., M.J.B. and D.S. oversaw research progress. S.E.B., T.A.P. and R.W. acquired and analyzed data for the fusilassin loop panels and individual fusilassin variants. S.E.B. performed all the bioinformatics, structural prediction, FusC cyclase engineering, FusC variant substrate scope testing and XgaC engineering. S.Y. performed and analyzed the MD simulations. P.J. and J.K.B. performed the MccJ25 cyclase engineering and integrin binding studies, with help from J.G.-N., G.C.M.d.C., C.R., B.K.O. and K.A. All authors performed data analysis. S.E.B. wrote the paper, with help from S.Y., editing and guidance from D.A.M. and M.J.B. and input from all other authors.

Corresponding authors

Correspondence to Mark J. Burk or Douglas A. Mitchell.

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Competing interests

M.J.B. and D.A.M. are co-founders of and own stock in Lassogen, Inc. S.E.B. and D.A.M. are inventors on a provisional patent application filed by the University of Illinois at Urbana-Champaign covering lasso cyclase engineering (US Provisional Application Ser. No. 63/673,853). M.J.B., P.A.J. and G.C.M.d.C. are inventors on a provisional patent application filed by Lassogen, Inc. for the design of lasso peptide integrin binders (US Provisional Application No. 63/612,957). The other authors declare no competing interests.

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Nature Chemical Biology thanks John Fetse, Sylvie Rebuffat and Jessica Swanson for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 FusA cationic residue substrate analysis.

a, MALDI-TOF-MS data of basic residues introduced into the loop of FusA via cell-free biosynthesis (CFB). The m/z values represent [M + H]+ for the cyclized lasso peptide. Endogenous proteases in the E. coli-derived lysate rapidly degrade linear peptides, so masses are typically only detected for the lasso peptide products. b, MALDI-TOF-MS time course of in vitro reconstitution reactions containing purified FusA I11R, FusB, FusC, and FusE. The [M + H]+ for the linear core peptide is indicated. The expected m/z for the lasso peptide is 2312 Da and is not detected. c, Arginine scan of the FusA core sequence. The sequence of fusilassin is provided (top) with the location of individual Arg variants denoted next to the corresponding spectrum: (black, treated with wild-type FusC; blue, treated with FusC R321G). Precursor peptide variants were generated and tested using CFB. The cyclized lasso peptide [M + H]+ mass is indicated next to the cyclized peak. The only difference in Arg tolerance is seen for positions 10–14.

Extended Data Fig. 2 Structural analysis of FusC.

a, Structure prediction comparison for FusC. Shown are the E. coli asparagine synthetase crystal structure (AsnB, PDB code: 1CT9)25 and FusC structures predicted by AlphaFold22, ESMFold23, and OmegaFold24. RMSD values (in Å) were calculated using the cealign function in PyMOL46. b, Residue conservation across the AlphaFold structure of FusC. Conservation was calculated using ConSurf45 where purple indicates highly conserved residues, green indicates highly variable residues, and yellow indicates insufficient data. ATP binding residues are shown with grey arrows and the hypervariable insertion region (labeled HVR), helix 11, and helix 18 are annotated. c, CASTp volume prediction of the FusC active site48. Sequence of FusC where blue highlighted letters indicate cavity lining residues.

Extended Data Fig. 3 Sequence similarity networks (SSNs) of lasso cyclases annotated by substrate electrostatic considerations.

The SSNs were constructed using EFI-EST27,42 on 7160 lasso cyclases identified by RODEO26 viewed at alignment score 120. The SSN is a full SSN (that is, not a repnode network) a, The SSN is colored based on the formal charge of the loop region of the cognate substrate. Loop charges were calculated only for the first 4 residues after the acceptor residue to ensure no tail residues were included in the analysis. b, Same as panel a, except the coloring indicates the formal charge of the ring region. Various NCBI accession codes used in the Extended Data Fig. 4 are provided as landmarks.

Source data

Extended Data Fig. 4 Lasso cyclase electrostatic surface analysis.

a, FusC-like cyclases and their corresponding core peptides are shown. Percent identities are in relation to FusC. b-d, Lasso cyclases corresponding to core peptides with extreme and opposite charged rings and loops. These are annotated in the SSNs in Extended Data Fig. 3. Charged residues in the ring and predicted loop are: blue (basic) and red (acidic). Loops were predicted based on the position of the most probable plug residue. Each electrostatic potential map was calculated using ChimeraX and the image depicts the backwall region of the cyclases (helix 11 in FusC). e, Alignment of FusC- and SnoC-like cyclases. FusC (WP_104612995.1), SnoC (AIA02568.1) and related cyclases are shown. Helix 11 is orange with the residues potentially contributing to loop specificity underlined and colored orange in each sequence (FusC S314 and R321). ATP-binding residues are shown in black bold. The core peptides associated with each cyclase are provided with the acceptor (red) and presumed plug (blue) indicated.

Extended Data Fig. 5 Molecular dynamics simulation snapshots and contact probability for FusA:FusC.

Snapshots from the MD simulation and contact heat plots are shown for: a, FusA with FusC; b, FusA I11R with FusC; c, FusA I11R with FusC R321G; d, FusA V13R with FusC; and e, FusA V13 with FusC R321G. The red text indicates variation from wild-type FusA/FusC. Snapshots were selected from the MD simulations based on high native contact value (Q > 0.8). The simulation data presented here can be obtained from the Dryad repository at: doi.org/10.5061/dryad.fttdz092h.

Extended Data Fig. 6 FusC R321 variant analysis.

a, MALDI-TOF-MS data for FusC wild-type, R321G, R321A, and R321D in vitro reconstitution reactions with all purified components. The cyclized lasso peptide [M + H]+ peak is orange and the mass is given. The linear core peak is blue. b, MALDI-TOF-MS analysis of CFB reactions expressing FusA variants with basic residues (Arg, Lys, His) at position 11, 12, and 13 Reactions were run with purified FusC R321G, FusB, and FusE. The [M + H]+ of the cyclized product is orange and the mass is given. c, Time course experiment comparing FusC WT and R321G with FusA. In vitro reconstitution reactions with all purified components were run, quenched with 60% MeCN (final) at the appropriate times, and analyzed with MALDI-TOF-MS. Orange: cyclized lasso peptide [M + H]+, blue: linear core peptide [M + H]+. d, Fraction of native contacts (Q) distributions. From MD simulations: orange, wild-type FusA with wild-type FusC (FusA:FusC); purple, FusA V13R with wild-type FusC (FusA V13R: FusC); blue, FusA V13R with FusC R321G (FusA V13R:FusC R321G). High probability density at a high fraction of native contacts correlates with the likelihood of successful lasso peptide formation. Bootstrapping (80% of the MD data over 200 iterations) was performed to calculate the probability density error. Data are presented as mean values ± standard deviation (SD). The simulation data presented here can be obtained from the Dryad repository at: doi.org/10.5061/dryad.fttdz092h.

Extended Data Fig. 7 Xanthomonin cyclase bioinformatic analysis and variant heterologous expression.

a, Xanthomonin biosynthetic gene cluster and precursor peptide sequences. Xanthomonin I and II have 7-residue rings, 4-residue loops, and tails that undergo proteolytic trimming during E. coli expression (portion in parentheses). b, Structural alignment of AlphaFold predicted FusC and XgaC (PyMOL cealignment). XgaC is tan while FusC is blue. The zoomed image depicts helix 11 of the superimposed structures. XgaC Lys324 and FusC Arg321 are shown as sticks. c, MALDI-TOF-MS data showing the results of the heterologous expression experiment to understand the loop-helix 11 interaction for xanthomonin II. The top two spectra are with wild-type XgaC while the bottom two spectra are with XgaC K324E. m/z 1293 is the wild-type cyclized xanthomonin II [M+Na]+ peak while m/z 1309 is the wild-type cyclized xanthomonin II [M + K]+ peak. The E8K variant is 1 Da lighter with [M+Na]+ at 1292 and [M + K]+ at 1308. d, Analysis of residue identity frequency at position 324 for cyclases from the xanthomonin II SSN group. Correlation of Lys at position 324 with Glu at position +1 is highlighted in blue. Correlation between large residues at position 324 with small +1 core peptide residues are highlighted in orange. This analysis used 224 XgaC homologs.

Extended Data Fig. 8 Microcin J25 cyclase bioinformatic analysis.

a, Structural alignment of AlphaFold predicted FusC and McjC (PyMOL cealignment). FusC is blue while McjC is tan. Zoom shows helix 11 FusC Arg321, McjC Lys252, and McjC Lys388 as sticks. b, Cartoon depiction and surface map for McjC highlighting a hole in the active site in the back wall region. The orange region contains McjC Lys252 and the lack of helix 11 creates a space large enough to peer through the predicted enzyme structure. c, Back wall regions for FusC, KleC, and UboC. FusC folds a lasso peptide with a 6-residue loop and contains a helix at the backwall (helix 11, yellow). KleC and UboC primarily display loops/coils at the back wall (yellow). These cyclases are responsible for folding lasso peptides with longer loops ( > 9 residues).

Extended Data Table 1 List of all variants tested in the loop 2×NNK panels organized by substrate and non-substrate
Full size table
Extended Data Table 2 FusC variants tested in CFB against basic FusA loop variants
Full size table

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figs. 1–17, Supplementary Tables 1–6 and Supplementary Data 1–6.

Reporting Summary

Supplementary Table 7

List of oligonucleotide sequences used in this study.

Source data

Source Data Fig. 1

Data used to generate the library heatmap.

Source Data Extended Data Fig. 3

RODEO-predicted dataset of lasso peptide substrate–lasso cyclase pairs.

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Barrett, S.E., Yin, S., Jordan, P. et al. Substrate interactions guide cyclase engineering and lasso peptide diversification. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01727-w

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