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 via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Newman, D.J. & Cragg, G.M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).
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).
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).
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).
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).
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).
Xie, L. et al. Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity. Science 303, 679–681 (2004).
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).
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).
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).
Mathavan, I. et al. Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides. Nat. Chem. Biol. 10, 340–342 (2014).
Tianero, M.D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl. Acad. Sci. USA 113, 1772–1777 (2016).
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).
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).
Philmus, B., Christiansen, G., Yoshida, W.Y. & Hemscheidt, T.K. Post-translational modification in microviridin biosynthesis. ChemBioChem 9, 3066–3073 (2008).
Zhao, G. et al. Structure and function of Escherichia coli RimK, an ATP-grasp fold, L-glutamyl ligase enzyme. Proteins 81, 1847–1854 (2013).
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).
Fawaz, M.V., Topper, M.E. & Firestine, S.M. The ATP-grasp enzymes. Bioorg. Chem. 39, 185–191 (2011).
Ouchi, T. et al. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus. Nat. Chem. Biol. 9, 277–283 (2013).
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).
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).
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).
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).
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).
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).
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).
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).
Koehnke, J. et al. Structural analysis of leader peptide binding enables leader-free cyanobactin processing. Nat. Chem. Biol. 11, 558–563 (2015).
Ortega, M.A. et al. Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB. Nature 517, 509–512 (2015).
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).
Dong, S.-H. et al. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold. eLife 4, e07607 (2015).
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).
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).
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).
Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Adams, P.D. Substructure search procedures for macromolecular structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 1966–1973 (2003).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
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).
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).
Dolinsky, T.J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522–W525 (2007).
Morris, G.M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
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
Authors and Affiliations
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
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
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.2200
This article is cited by
-
Phylogenomic analysis of the diversity of graspetides and proteins involved in their biosynthesis
Biology Direct (2022)
-
Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide
Nature Chemistry (2022)
-
The core of the matter
Nature Chemical Biology (2021)
-
Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis
Nature Communications (2021)
-
Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB
Nature Chemical Biology (2021)