Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

Abstract

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Biosynthesis of microviridin J.
Figure 2: Binding and cyclization activity of macrocyclases toward MdnA variants.
Figure 3: Overall structures of MdnC and MdnB.
Figure 4: MdnC uses ATP to catalyze the macrocyclization of the precursor peptide MdnA.
Figure 5: Leader-peptide-directed peptide macrocyclization in the microviridin J biosynthetic pathway.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Newman, D.J. & Cragg, G.M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

    CAS  Article  Google Scholar 

  2. 2

    Harvey, A.L., Edrada-Ebel, R. & Quinn, R.J. The re-emergence of natural products for drug discovery in the genomics era. Nat. Rev. Drug Discov. 14, 111–129 (2015).

    CAS  Article  Google Scholar 

  3. 3

    McIntosh, J.A., Donia, M.S. & Schmidt, E.W. Ribosomal peptide natural products: bridging the ribosomal and nonribosomal worlds. Nat. Prod. Rep. 26, 537–559 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Arnison, P.G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Ortega, M.A. & van der Donk, W.A. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products. Cell Chem. Biol. 23, 31–44 (2016).

    CAS  Article  Google Scholar 

  6. 6

    Crone, W.J.K., Leeper, F.J. & Truman, A.W. Identification and characterisation of the gene cluster for the anti-MRSA antibiotic bottromycin: expanding the biosynthetic diversity of ribosomal peptides. Chem. Sci. (Camb.) 3, 3516 (2012).

    CAS  Article  Google Scholar 

  7. 7

    Xie, L. et al. Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity. Science 303, 679–681 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Oman, T.J. & van der Donk, W.A. Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nat. Chem. Biol. 6, 9–18 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Pan, S.J. & Link, A.J. Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions. J. Am. Chem. Soc. 133, 5016–5023 (2011).

    CAS  Article  Google Scholar 

  10. 10

    Weiz, A.R. et al. Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions. Angew. Chem. Int. Ed. Engl. 53, 3735–3738 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Mathavan, I. et al. Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides. Nat. Chem. Biol. 10, 340–342 (2014).

    CAS  Article  Google Scholar 

  12. 12

    Tianero, M.D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl. Acad. Sci. USA 113, 1772–1777 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angew. Chem. Int. Ed. Engl. 47, 7756–7759 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Weiz, A.R. et al. Leader peptide and a membrane protein scaffold guide the biosynthesis of the tricyclic peptide microviridin. Chem. Biol. 18, 1413–1421 (2011).

    CAS  Article  Google Scholar 

  15. 15

    Philmus, B., Christiansen, G., Yoshida, W.Y. & Hemscheidt, T.K. Post-translational modification in microviridin biosynthesis. ChemBioChem 9, 3066–3073 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Zhao, G. et al. Structure and function of Escherichia coli RimK, an ATP-grasp fold, L-glutamyl ligase enzyme. Proteins 81, 1847–1854 (2013).

    CAS  Article  Google Scholar 

  17. 17

    Iyer, L.M., Abhiman, S., Maxwell Burroughs, A. & Aravind, L. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins. Mol. Biosyst. 5, 1636–1660 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Fawaz, M.V., Topper, M.E. & Firestine, S.M. The ATP-grasp enzymes. Bioorg. Chem. 39, 185–191 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Ouchi, T. et al. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus. Nat. Chem. Biol. 9, 277–283 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Liu, S. et al. Allosteric inhibition of Staphylococcus aureus D-alanine:D-alanine ligase revealed by crystallographic studies. Proc. Natl. Acad. Sci. USA 103, 15178–15183 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Wang, H., Falck, J.R., Hall, T.M.T. & Shears, S.B. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat. Chem. Biol. 8, 111–116 (2011).

    Article  Google Scholar 

  22. 22

    Rohrlack, T., Christoffersen, K., Kaebernick, M. & Neilan, B.A. Cyanobacterial protease inhibitor microviridin J causes a lethal molting disruption in Daphnia pulicaria. Appl. Environ. Microbiol. 70, 5047–5050 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Liu, Y., Zheng, T. & Bruner, S.D. Structural basis for phosphopantetheinyl carrier domain interactions in the terminal module of nonribosomal peptide synthetases. Chem. Biol. 18, 1482–1488 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Koehnke, J. et al. The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat. Struct. Mol. Biol. 19, 767–772 (2012).

    CAS  Article  Google Scholar 

  25. 25

    Wang, B., Zhao, A., Novick, R.P. & Muir, T.W. Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence. Proc. Natl. Acad. Sci. USA 112, 10679–10684 (2015).

    CAS  Article  Google Scholar 

  26. 26

    Pan, S.J., Rajniak, J., Cheung, W.L. & Link, A.J. Construction of a single polypeptide that matures and exports the lasso peptide microcin J25. ChemBioChem 13, 367–370 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Oman, T.J., Knerr, P.J., Bindman, N.A., Velásquez, J.E. & van der Donk, W.A. An engineered lantibiotic synthetase that does not require a leader peptide on its substrate. J. Am. Chem. Soc. 134, 6952–6955 (2012).

    CAS  Article  Google Scholar 

  28. 28

    Koehnke, J. et al. Structural analysis of leader peptide binding enables leader-free cyanobactin processing. Nat. Chem. Biol. 11, 558–563 (2015).

    CAS  Article  Google Scholar 

  29. 29

    Ortega, M.A. et al. Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB. Nature 517, 509–512 (2015).

    CAS  Article  Google Scholar 

  30. 30

    Burkhart, B.J., Hudson, G.A., Dunbar, K.L. & Mitchell, D.A. A prevalent peptide-binding domain guides ribosomal natural product biosynthesis. Nat. Chem. Biol. 11, 564–570 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Dong, S.-H. et al. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold. eLife 4, e07607 (2015).

    Article  Google Scholar 

  32. 32

    Cheung, W.L., Pan, S.J. & Link, A.J. Much of the microcin J25 leader peptide is dispensable. J. Am. Chem. Soc. 132, 2514–2515 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Philmus, B., Guerrette, J.P. & Hemscheidt, T.K. Substrate specificity and scope of MvdD, a GRASP-like ligase from the microviridin biosynthetic gene cluster. ACS Chem. Biol. 4, 429–434 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Schmidt, E.W. et al. Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci. USA 102, 7315–7320 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Adams, P.D. Substructure search procedures for macromolecular structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 1966–1973 (2003).

  38. 38

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  39. 39

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  40. 40

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  41. 41

    Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  Article  Google Scholar 

  42. 42

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  Google Scholar 

  43. 43

    Goldenberg, O., Erez, E., Nimrod, G. & Ben-Tal, N. The ConSurf-DB: pre-calculated evolutionary conservation profiles of protein structures. Nucleic Acids Res. 37, D323–D327 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    CAS  Article  Google Scholar 

  45. 45

    Dolinsky, T.J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522–W525 (2007).

    Article  Google Scholar 

  46. 46

    Morris, G.M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the staff at the 21-ID-G and 22-ID beamlines at Argonne National Laboratory Advanced Photon Source for help with data acquisition and processing. We thank T. Montavon and S. Lagishetty for providing experimental contributions supporting this work. We also are grateful to N. Polfer and A. Patrick for assistance with peptide synthesis. This work was supported by US National Institutes of Health grant GM086570 and funds from the University of Florida (S.D.B.).

Author information

Affiliations

Authors

Contributions

K.L., H.L.C. and S.D.B. conceived the project and designed the experiments. K.L. and H.L.C. performed cloning, expression screening, protein purification, crystallization and X-ray diffraction data collection, structure solution and refinement. H.L.C. carried out kinetic analysis. K.L. and G.L. prepared and analyzed mutant proteins. K.L., H.L.C., Y.D. and S.D.B. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Steven D Bruner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–13. (PDF 1195 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, K., Condurso, H., Li, G. et al. Structural basis for precursor protein–directed ribosomal peptide macrocyclization. Nat Chem Biol 12, 973–979 (2016). https://doi.org/10.1038/nchembio.2200

Download citation

Further reading

Search

Quick links