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Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

Nature volume 454, pages 903–906 (2008)Cite this article

Abstract

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties1. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations2. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

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Figure 1: Summary of enterobactin synthesis.
Figure 2: Structure of the EntF T–TE fragment.
Figure 3: Interaction with Sfp.
Figure 4: Interaction with the C domain.

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Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the Protein Data Bank under accession code 2ROQ.

References

  1. Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L. O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007)

    Article  CAS  Google Scholar 

  3. Gehring, A. M., Bradley, K. A. & Walsh, C. T. Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate. Biochemistry 36, 8495–8503 (1997)

    Article  CAS  Google Scholar 

  4. Gehring, A. M., Mori, I. & Walsh, C. T. Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochemistry 37, 2648–2659 (1998)

    Article  CAS  Google Scholar 

  5. Wagner, G. The importance of being floppy. Nature Struct. Biol. 2, 255–257 (1995)

    Article  CAS  Google Scholar 

  6. Koglin, A. et al. Conformational switches modulate protein interactions in peptide antibiotic synthetases. Science 312, 273–276 (2006)

    Article  ADS  CAS  Google Scholar 

  7. Weber, T., Baumgartner, R., Renner, C., Marahiel, M. A. & Holak, T. A. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases. Structure 8, 407–418 (2000)

    Article  CAS  Google Scholar 

  8. Findlow, S. C., Winsor, C., Simpson, T. J., Crosby, J. & Crump, M. P. Solution structure and dynamics of oxytetracycline polyketide synthase acyl carrier protein from Streptomyces rimosus . Biochemistry 42, 8423–8433 (2003)

    Article  CAS  Google Scholar 

  9. Johnson, M. A., Peti, W., Herrmann, T., Wilson, I. A. & Wuthrich, K. Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC 7120 with a variant phosphopantetheinylation-site sequence. Protein Sci. 15, 1030–1041 (2006)

    Article  CAS  Google Scholar 

  10. Zornetzer, G. A., Fox, B. G. & Markley, J. L. Solution structures of spinach acyl carrier protein with decanoate and stearate. Biochemistry 45, 5217–5227 (2006)

    Article  CAS  Google Scholar 

  11. Kim, Y. & Prestegard, J. H. A dynamic model for the structure of acyl carrier protein in solution. Biochemistry 28, 8792–8797 (1989)

    Article  CAS  Google Scholar 

  12. Bruner, S. D. et al. Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE. Structure 10, 301–310 (2002)

    Article  CAS  Google Scholar 

  13. Samel, S. A., Wagner, B., Marahiel, M. A. & Essen, L. O. The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-ribosomal lipopeptide. J. Mol. Biol. 359, 876–889 (2006)

    Article  CAS  Google Scholar 

  14. Koglin, A. et al. Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 10.1038/nature07161 (this issue)

  15. Leibundgut, M., Jenni, S., Frick, C. & Ban, N. Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science 316, 288–290 (2007)

    Article  ADS  CAS  Google Scholar 

  16. Lomakin, I. B., Xiong, Y. & Steitz, T. A. The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together. Cell 129, 319–332 (2007)

    Article  Google Scholar 

  17. Maier, T., Jenni, S. & Ban, N. Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science 311, 1258–1262 (2006)

    Article  ADS  CAS  Google Scholar 

  18. Jenni, S., Leibundgut, M., Maier, T. & Ban, N. Architecture of a fungal fatty acid synthase at 5 Å resolution. Science 311, 1263–1267 (2006)

    Article  ADS  CAS  Google Scholar 

  19. Jenni, S. et al. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science 316, 254–261 (2007)

    Article  ADS  CAS  Google Scholar 

  20. Lambalot, R. H. et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 3, 923–936 (1996)

    Article  CAS  Google Scholar 

  21. Lambalot, R. H. & Walsh, C. T. Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J. Biol. Chem. 270, 24658–24661 (1995)

    Article  CAS  Google Scholar 

  22. Roche, E. D. & Walsh, C. T. Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. Biochemistry 42, 1334–1344 (2003)

    Article  CAS  Google Scholar 

  23. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wuthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl Acad. Sci. USA 95, 13585–13590 (1998)

    Article  ADS  CAS  Google Scholar 

  24. Rovnyak, D. et al. Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J. Magn. Reson. 170, 15–21 (2004)

    Article  ADS  CAS  Google Scholar 

  25. Frueh, D. P. et al. Non-uniformly sampled double-TROSY hNcaNH experiments for NMR sequential assignments of large proteins. J. Am. Chem. Soc. 128, 5757–5763 (2006)

    Article  CAS  Google Scholar 

  26. Frueh, D. P., Vosburg, D. A., Walsh, C. T. & Wagner, G. Determination of all NOes in 1H–13C-Me-ILV-U-2H–15N proteins with two time-shared experiments. J. Biomol. NMR 34, 31–40 (2006)

    Article  CAS  Google Scholar 

  27. Guntert, P., Mumenthaler, C. & Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997)

    Article  CAS  Google Scholar 

  28. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997)

    Article  ADS  CAS  Google Scholar 

  29. Tolman, J. R., Flanagan, J. M., Kennedy, M. A. & Prestegard, J. H. Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc. Natl Acad. Sci. USA 92, 9279–9283 (1995)

    Article  ADS  CAS  Google Scholar 

  30. Gardner, K. H. & Kay, L. E. Production and incorporation of 15N, 13C, 2H (1H-δ1 methyl) isoleucine into proteins for multidimensional NMR studies. J. Am. Chem. Soc. 119, 7599–7600 (1997)

    Article  CAS  Google Scholar 

  31. Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E. & Dahlquist, F. W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 177, 44–73 (1989)

    Article  CAS  Google Scholar 

  32. Ferentz, A. E. & Wagner, G. NMR spectroscopy: a multifaceted approach to macromolecular structure. Q. Rev. Biophys. 33, 29–65 (2000)

    Article  CAS  Google Scholar 

  33. Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999)

    Article  CAS  Google Scholar 

  34. Hyberts, S. G. & Wagner, G. IBIS – a tool for automated sequential assignment of protein spectra from triple resonance experiments. J. Biomol. NMR 26, 335–344 (2003)

    Article  CAS  Google Scholar 

  35. Pervushin, K., Riek, R., Wider, G. & Wuthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997)

    Article  ADS  CAS  Google Scholar 

  36. Pervushin, K. & Eletsky, A. A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment. J. Biomol. NMR 25, 147–152 (2003)

    Article  CAS  Google Scholar 

  37. Grzesiek, S., Anglister, J. & Bax, A. Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J. Magn. Reson. B. 101, 114–119 (1993)

    Article  CAS  Google Scholar 

  38. Bax, A., Clore, M. & Gronenborn, A. M. 1H-1H Correlation via isotropic mixing of 13C magentization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J. Magn. Reson. 88, 425–431 (1990)

    ADS  CAS  Google Scholar 

  39. Ikura, M., Kay, L. E. & Bax, A. Improved three-dimensional 1H-13C-1H correlation spectroscopy of a 13C-labeled protein using constant-time evolution. J. Biomol. NMR 1, 299–304 (1991)

    Article  CAS  Google Scholar 

  40. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

    Article  CAS  Google Scholar 

  41. Medek, A., Olejniczak, E. T., Meadows, R. P. & Fesik, S. W. An approach for high-throughput structure determination of proteins by NMR spectroscopy. J. Biomol. NMR 18, 229–238 (2000)

    Article  CAS  Google Scholar 

  42. Delaglio, F. et al. NMRPipe a Multidimensional Spectra Processing System Based on UNIX Pipes. J. Biomol. NMR 6, 277–293 (1995)

    Article  CAS  Google Scholar 

  43. Keller, R. L. J. The Computer Aided Resonance Assignment Tutorial (Cantina, 2004)

    Google Scholar 

  44. Schwede, T., Kopp, J., Guex, N. & Peitsch, M. C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003)

    Article  CAS  Google Scholar 

  45. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

    Article  CAS  Google Scholar 

  46. Nederveen, A. J. et al. RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank. Proteins 59, 662–672 (2005)

    Article  CAS  Google Scholar 

  47. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Sastry and A. Marintchev for advice in sample preparation; Z. Zhou for providing the Ser48Ala plasmid; Z.-Y. Sun for participating in the development of new NMR techniques; and P. Selenko for comments on the manuscript. This work was supported by NIH grants GM47467, EB 002026, and GM066360 and a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund (D.A.V.). A.K. thanks the Human Frontier Science Program for a long-term fellowship awarded in April 2007.

Author Contributions D.P.F., G.W. and C.T.W. designed research and wrote the manuscript. D.P.F. conducted the research including protein expression, resonance assignment and structure calculation. H.A. prepared various samples and helped during the acquisition of NMR experiments. D.A.V. prepared various samples. A.K. helped during the structure calculation and prepared the C domains EntD and Sfp samples. A.E.B. participated in early stages of the project.

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  1. David A. Vosburg

    Present address: Present address: Department of Chemistry, Harvey Mudd College, 301 Platt Boulevard, Claremont, California 91711-5901, USA.,

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA,

    Dominique P. Frueh, Haribabu Arthanari, Alexander Koglin, David A. Vosburg, Andrew E. Bennett, Christopher T. Walsh & Gerhard Wagner

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Correspondence to Dominique P. Frueh, Christopher T. Walsh or Gerhard Wagner.

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Frueh, D., Arthanari, H., Koglin, A. et al. Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase. Nature 454, 903–906 (2008). https://doi.org/10.1038/nature07162

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