Skip to main content

Thank you for visiting 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.

Structures of two distinct conformations of holo-non-ribosomal peptide synthetases


Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs)1,2,3,4. During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion5. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Ribbon diagrams of complete NRPS modules.
Figure 2: NRPS domain structures.
Figure 3: Conformational dynamics in NRPS modules.
Figure 4: Dynamics of the NRPS cycle.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession numbers 4ZXH (holo-AB3403), 4ZXI (holo-AB3403 bound to AMP and glycine), and 4ZXJ (holo-EntF bound to Ser-AVS).


  1. Koglin, A. & Walsh, C. T. Structural insights into nonribosomal peptide enzymatic assembly lines. Nat. Prod. Rep. 26, 987–1000 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Fischbach, M. A. & Walsh, C. T. Antibiotics for emerging pathogens. Science 325, 1089–1093 (2009)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  3. Walsh, C. T. The chemical versatility of natural-product assembly lines. Acc. Chem. Res. 41, 4–10 (2008)

    CAS  PubMed  Google Scholar 

  4. Walsh, C. T. & Fischbach, M. A. Natural products version 2.0: connecting genes to molecules. J. Am. Chem. Soc. 132, 2469–2493 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Strieker, M., Tanovic´, A. & Marahiel, M. A. Nonribosomal peptide synthetases: structures and dynamics. Curr. Opin. Struct. Biol. 20, 234–240 (2010)

    CAS  PubMed  Google Scholar 

  6. Mercer, A. C. & Burkart, M. D. The ubiquitous carrier protein--a window to metabolite biosynthesis. Nat. Prod. Rep. 24, 750–773 (2007)

    CAS  PubMed  Google Scholar 

  7. Gulick, A. M. Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem. Biol. 4, 811–827 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Conti, E., Stachelhaus, T., Marahiel, M. A. & Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Mitchell, C. A., Shi, C., Aldrich, C. C. & Gulick, A. M. Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains. Biochemistry 51, 3252–3263 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sundlov, J. A., Shi, C., Wilson, D. J., Aldrich, C. C. & Gulick, A. M. Structural and functional investigation of the intermolecular interaction between NRPS adenylation and carrier protein domains. Chem. Biol. 19, 188–198 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Goodrich, A. C., Harden, B. J. & Frueh, D. P. Solution structure of a nonribosomal peptide synthetase carrier protein loaded with its substrate reveals transient, well-defined contacts. J. Am. Chem. Soc. 137, 12100–12109 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Jaremko, M. J., Lee, D. J., Opella, S. J. & Burkart, M. D. Structure and substrate sequestration in the pyoluteorin type II peptidyl carrier protein PltL. J. Am. Chem. Soc. 137, 11546–11549 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Tanovic, A., Samel, S. A., Essen, L. O. & Marahiel, M. A. Crystal structure of the termination module of a nonribosomal peptide synthetase. Science 321, 659–663 (2008)

    CAS  PubMed  ADS  Google Scholar 

  14. Clemmer, K. M., Bonomo, R. A. & Rather, P. N. Genetic analysis of surface motility in Acinetobacter baumannii. Microbiology 157, 2534–2544 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Rumbo-Feal, S. et al. Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in biofilm compared to planktonic cells. PLoS One 8, e72968 (2013)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  16. Giles, S. K., Stroeher, U. H., Eijkelkamp, B. A. & Brown, M. H. Identification of genes essential for pellicle formation in Acinetobacter baumannii. BMC Microbiol . 15, 116 (2015)

    PubMed  PubMed Central  Google Scholar 

  17. Bloudoff, K., Rodionov, D. & Schmeing, T. M. Crystal structures of the first condensation domain of CDA synthetase suggest conformational changes during the synthetic cycle of nonribosomal peptide synthetases. J. Mol. Biol. 425, 3137–3150 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Marahiel, M. A., Stachelhaus, T. & Mootz, H. D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997)

    CAS  PubMed  Google Scholar 

  19. Frueh, D. P. et al. Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase. Nature 454, 903–906 (2008)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  20. 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  PubMed  PubMed Central  Google Scholar 

  21. Qiao, C., Wilson, D. J., Bennett, E. M. & Aldrich, C. C. A mechanism-based aryl carrier protein/thiolation domain affinity probe. J. Am. Chem. Soc. 129, 6350–6351 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Miller, B. R., Sundlov, J. A., Drake, E. J., Makin, T. A. & Gulick, A. M. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases. Proteins 82, 2691–2702 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Gaudelli, N. M., Long, D. H. & Townsend, C. A. β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis. Nature 520, 383–387 (2015)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  24. Maruyama, C. et al. A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis. Nature Chem. Biol. 8, 791–797 (2012)

    CAS  Google Scholar 

  25. Dutta, S. et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  26. Adams, M. D. et al. Comparative genome sequence analysis of multidrug-resistant Acinetobacter baumannii. J. Bacteriol. 190, 8053–8064 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001)

    CAS  PubMed  Google Scholar 

  28. Doublié, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997)

    PubMed  Google Scholar 

  29. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011)

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  31. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171–1179 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

  33. Urzhumtsev, A., Afonine, P. V. & Adams, P. D. TLS from fundamentals to practice. Crystallogr. Rev . 19, 230–270 (2013)

    PubMed  PubMed Central  Google Scholar 

  34. 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)

    CAS  PubMed  Google Scholar 

  35. Luft, J. R. et al. A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol. 142, 170–179 (2003)

    CAS  PubMed  Google Scholar 

  36. Sundlov, J. A. & Gulick, A. M. Structure determination of the functional domain interaction of a chimeric nonribosomal peptide synthetase from a challenging crystal with noncrystallographic translational symmetry. Acta Crystallogr. D 69, 1482–1492 (2013)

    CAS  PubMed  Google Scholar 

  37. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    PubMed  Google Scholar 

  38. Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification - powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    CAS  PubMed  Google Scholar 

  40. Yang, Z., Fang, J., Chittuluru, J., Asturias, F. J. & Penczek, P. A. Iterative stable alignment and clustering of 2D transmission electron microscope images. Structure 20, 237–247 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Ikeuchi, H., Meyer, M. E., Ding, Y., Hiratake, J. & Richards, N. G. A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase. Bioorg. Med. Chem. 17, 6641–6650 (2009)

    CAS  PubMed  Google Scholar 

  42. Rusnak, F., Faraci, W. S. & Walsh, C. T. Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylate product. Biochemistry 28, 6827–6835 (1989)

    CAS  PubMed  Google Scholar 

  43. Horswill, A. R. & Escalante-Semerena, J. C. Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis. Biochemistry 41, 2379–2387 (2002)

    CAS  PubMed  Google Scholar 

  44. Reger, A. S., Carney, J. M. & Gulick, A. M. Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase. Biochemistry 46, 6536–6546 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Wilson, D. J. & Aldrich, C. C. A continuous kinetic assay for adenylation enzyme activity and inhibition. Anal. Biochem. 404, 56–63 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Reuter, D. C., McIntosh, J. E., Guinn, A. C. & Madera, A. M. Synthesis of vinyl sulfonamides using the Horner reaction. Synthesis 2003, 2321–2324 (2003)

    Google Scholar 

Download references


We thank R. Sanishvili for assistance with data collection. This work was funded in part by National Institutes of Health GM-068440 (to A.M.G.) and GM-115601 (to G.S.), and Award W81XWH-11-2-0218 from the Telemedicine and Advanced Technology Research Center of the US Army Medical Research and Materiel Command (A.M.G.). Data were collected at the GM/CA beamline of the Advanced Photon Source, which is funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006) under Department of Energy contract number DE-AC02-06CH11357 to A.P.S. A Stafford Fellowship (to B.R.M.) and support from the Hauptman-Woodward Institute is acknowledged.

Author information

Authors and Affiliations



C.L.A. characterized activity of and initially crystallized AB3403. J.A.S. initially crystallized EntF. E.J.D. and B.R.M. optimized crystal, and solved and refined the models of AB3403 and EntF, respectively. C.S. and C.C.A. designed and synthesized the mechanism-based inhibitor. J.T.T. and G.S. performed and analysed the single-particle electron microscopy. A.M.G., E.J.D., B.R.M., G.S., J.T.T., C.C.A., and C.S. analysed the results and wrote the manuscript. All authors saw and approved the manuscript.

Corresponding author

Correspondence to Andrew M. Gulick.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Structure-based alignment of EntF, AB3403, and SrfA-C.

Condensation, adenylation, PCP, and thioesterase domains are represented with bars in grey, pink, green–cyan, and blue. Conserved motifs and catalytically important residues are highlighted with the same colours, including the HHxxxD motif of the condensation domains, the aspartic acid hinge that separates the N- and C-terminal subdomains of the adenylation domain, the GGHS motif that is the site of pantetheinylation in the PCP, and the catalytic nucleophile of the thioesterase domain. The SrfA-C, AB3403, and EntF proteins share approximately 26% sequence identity. The adenylation and PCP domains are more well-conserved, sharing ~35% identity, whereas the condensation (21%) and thioesterase (25%) domains are less well conserved. Domain boundaries are described in the table below.

Extended Data Figure 2 Substrate specificity of full-length AB3403.

Amino-acid specificity of AB3403 was recorded for all 20 proteinogenic amino acids, as well as 4-chlorobenzoate (4CB) and 4-hydroxybenzoate (4HB). Average values and standard deviations are shown for three replicates with each substrate; results were recorded as micromoles of radiolabelled ATP incorporated per minute per milligram of enzyme. Apparent kinetic constants are also shown for ATP and glycine calculated from duplicate measurements for four to six substrate concentrations.

Extended Data Figure 3 Stereo representations of electron density figures shown in Fig. 2.

To better visualize the active sites and electron density quality, stereo figures are included in the extended data. In all panels, density is shown with coefficients of the form (Fo − Fc) calculated before inclusion of ligands and contoured at 3σ. a, Stereo representation of electron density of AB3403 condensation domain shows the phosphopantethine on Ser1006 approaching His145 within the condensation domain pocket. Inhibitor carbon atoms in green, carbons of residues within 5 Å of inhibitor in grey, nitrogen in blue, oxygen in red, sulphur in yellow, and water in light blue. b, Electron density of the nucleotide binding pocket of AB3403 bound to glycine and AMP. Stereo representation of electron density shows the AMP, glycine, and Mg+ present in the active site of the adenylation domain. Ligand carbon atoms are in green, carbons of residues within 5 Å of inhibitor in grey, nitrogen in blue, oxygen in red, phosphorus in orange, and the Mg+ cofactor in purple. c, Stereo representation of the electron density shows the phosphopantethine on Ser1006 covalently attached to the Ser-AVS inhibitor in the active site of the adenylation domain. Inhibitor carbon atoms in green, carbons of residues within 4 Å of inhibitor in grey, nitrogen in blue, oxygen in red, phosphorus in orange, sulphur in yellow, and water in light blue.

Extended Data Figure 4 Comparison of AB3403 and SrfA-C PCP-condensation domain interaction.

Stereo representation illustrating different orientations of the PCP domains of SrfA-C and AB3403 relative to the condensation domains with which they interact. AB3403 is shown with a white condensation domain and a green-cyan PCP. SrfA-C is shown with a yellow condensation domain and a pale blue PCP. The pantetheine of AB3403 is shown bound to Ser1006. The position of Ser1003, mutated to an alanine residue in SrfA-C, is also highlighted.

Extended Data Figure 5 Comparison of AB3403 thioesterase domain to the functional PCP–thioesterase interaction.

Stereo representation of the thioesterase (blue) domain of AB3403 interacts with the back face of the PCP domain in AB3403. The functional interaction between the EntF thioesterase domain and its holo-PCP, trapped crystallographically, illustrates that the same face of the thioesterase domain interacts functionally (PDB 3TEJ). A 28-residue insertion of AB3403 is coloured yellow.

Extended Data Figure 6 Synthesis of Ser-AVS.

The Ser-AVS probe was synthesized following similar protocols described elsewhere41,46. Garner’s aldehyde 1 was coupled with 2 using LiHMDS to exclusively furnish the (E)-vinylsulfonamide 3. Mitsunobu coupling of 3 with bis-Boc adenosine 4 afforded 5, which was globally deprotected using 80% aqueous trifluoroacetic acid to yield Ser-AVS.

Extended Data Figure 7 Electrophoretic mobility of EntF.

a, Native gel electrophoresis. Lane 1: EntF. Lane2: EntF incubated with fourfold molar excess of Ser-AVS inhibitor. Lane 3: EntF Crystals. Lane 4: novex NativeMark labelled in kilodaltons. b, Denaturing gel electrophoresis using loading buffer with SDS and β-mercaptoethanol. Gel lane 1: EntF. Lane 2: EntF incubated four times with Ser-AVS inhibitor. Lane 3: Life Technologies Mark12 labelled in kilodaltons. The native gel shows the inhibited EntF in a more compact conformation compared with EntF without the inhibitor.

Extended Data Figure 8 Negative-stain electron microscopy analysis of EntF.

a, Raw electron microscopy image of negative-stained EntF. b, Class averages of EntF particles.

Extended Data Table 1 Diffraction data statistics and refinement statistics for AB3403
Extended Data Table 2 Diffraction data statistics and refinement statistics for EntF

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion. (PDF 114 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Drake, E., Miller, B., Shi, C. et al. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529, 235–238 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing