[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
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.

  • Letter
  • Published:

Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase

Nature volume 529, pages 239–242 (2016)Cite this article

Subjects

Abstract

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics1. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites2,3. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space3,4. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase5,6. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A schematic of the action of the linear gramicidin synthetase initiation module.
Figure 2: Crystal structures representing the steps of the synthesis cycle in the LgrA initiation module.
Figure 3: Interdomain interfaces of the initiation module.
Figure 4: Comparisons of the F domain to sugar and tRNA formyltransferases.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Crystallography data have been deposited in the Protein Data Bank under accession codes 5ES5, 5ES6, 5ES7, 5ES8 and 5ES9.

References

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

    ADS  CAS  PubMed  Google Scholar 

  2. Weissman, K. J. The structural biology of biosynthetic megaenzymes. Nature Chem. Biol. 11, 660–670 (2015)

    CAS  Google Scholar 

  3. Hur, G. H., Vickery, C. R. & Burkart, M. D. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat. Prod. Rep. 29, 1074–1098 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Walsh, C. T. et al. Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Curr. Opin. Chem. Biol. 5, 525–534 (2001)

    CAS  PubMed  Google Scholar 

  5. Kessler, N., Schuhmann, H., Morneweg, S., Linne, U. & Marahiel, M. A. The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains. J. Biol. Chem. 279, 7413–7419 (2004)

    CAS  PubMed  Google Scholar 

  6. Schoenafinger, G., Schracke, N., Linne, U. & Marahiel, M. A. Formylation domain: an essential modifying enzyme for the nonribosomal biosynthesis of linear gramicidin. J. Am. Chem. Soc. 128, 7406–7407 (2006)

    CAS  PubMed  Google Scholar 

  7. Lawen, A. & Zocher, R. Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described. J. Biol. Chem. 265, 11355–11360 (1990)

    CAS  PubMed  Google Scholar 

  8. Robbel, L. & Marahiel, M. A. Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J. Biol. Chem. 285, 27501–27508 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Konz, D., Klens, A., Schorgendorfer, K. & Marahiel, M. A. The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases. Chem. Biol. 4, 927–937 (1997)

    CAS  PubMed  Google Scholar 

  10. Cheng, Y. Q. Deciphering the biosynthetic codes for the potent anti-SARS-CoV cyclodepsipeptide valinomycin in Streptomyces tsusimaensis ATCC 15141. ChemBioChem 7, 471–477 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Schneider, T. L., Shen, B. & Walsh, C. T. Oxidase domains in epothilone and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation. Biochemistry 42, 9722–9730 (2003)

    CAS  PubMed  Google Scholar 

  12. Koketsu, K., Minami, A., Watanabe, K., Oguri, H. & Oikawa, H. Pictet-Spenglerase involved in tetrahydroisoquinoline antibiotic biosynthesis. Curr. Opin. Chem. Biol. 16, 142–149 (2012)

    CAS  PubMed  Google Scholar 

  13. Schmitt, E., Panvert, M., Blanquet, S. & Mechulam, Y. Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet. EMBO J. 17, 6819–6826 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Almassy, R. J., Janson, C. A., Kan, C. C. & Hostomska, Z. Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase. Proc. Natl Acad. Sci. USA 89, 6114–6118 (1992)

    ADS  CAS  PubMed  Google Scholar 

  15. Thoden, J. B., Goneau, M. F., Gilbert, M. & Holden, H. M. Structure of a sugar N-formyltransferase from Campylobacter jejuni. Biochemistry 52, 6114–6126 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Wallace, B. A. Common structural features in gramicidin and other ion channels. Bioessays 22, 227–234 (2000)

    CAS  PubMed  Google Scholar 

  17. 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 

  18. Yonus, H. et al. Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains. J. Biol. Chem. 283, 32484–32491 (2008)

    CAS  PubMed  Google Scholar 

  19. Reger, A. S., Wu, R., Dunaway-Mariano, D. & Gulick, A. M. Structural characterization of a 140 degrees domain movement in the two-step reaction catalyzed by 4-chlorobenzoate:CoA ligase. Biochemistry 47, 8016–8025 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 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 

  21. 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 

  22. 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)

    ADS  CAS  PubMed  Google Scholar 

  23. Liu, Y. & Bruner, S. D. Rational manipulation of carrier-domain geometry in nonribosomal peptide synthetases. ChemBioChem 8, 617–621 (2007)

    CAS  PubMed  Google Scholar 

  24. Rouhiainen, L. et al. Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90. Mol. Microbiol. 37, 156–167 (2000)

    CAS  PubMed  Google Scholar 

  25. Lohman, J. R. et al. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins. Proteins 82, 1210–1218 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Bode, H. B. et al. Structure elucidation and activity of kolossin A, the d-/l-pentadecapeptide product of a giant nonribosomal peptide synthetase. Angew. Chem. Int. Ed. Engl. 54, 10352–10355 (2015)

    CAS  PubMed  Google Scholar 

  27. Zhao, C. et al. Oxazolomycin biosynthesis in Streptomyces albus JA3453 featuring an “acyltransferase-less” type I polyketide synthase that incorporates two distinct extender units. J. Biol. Chem. 285, 20097–20108 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Clardy, J., Fischbach, M. A. & Walsh, C. T. New antibiotics from bacterial natural products. Nature Biotechnol. 24, 1541–1550 (2006)

    CAS  Google Scholar 

  29. Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hosaka, T. et al. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12. Nature Biotechnol. 27, 462–464 (2009)

    CAS  Google Scholar 

  31. Nazi, I., Koteva, K. P. & Wright, G. D. One-pot chemoenzymatic preparation of coenzyme A analogues. Anal. Biochem. 324, 100–105 (2004)

    CAS  PubMed  Google Scholar 

  32. Acerbo, A. S., Cook, M. J. & Gillilan, R. E. Upgrade of MacCHESS facility for X-ray scattering of biological macromolecules in solution. J. Synchrotron Radiat. 22, 180–186 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Skou, S., Gillilan, R. E. & Ando, N. Synchrotron-based small-angle X-ray scattering of proteins in solution. Nature Protocols 9, 1727–1739 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Nielsen, S. S. et al. BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis. J. Appl. Crystallogr. 42, 959–964 (2009)

    CAS  Google Scholar 

  35. Svergun, D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992)

    CAS  Google Scholar 

  36. Franke, D. & Svergun, D. I. DAMMIF, a program for rapidab-initioshape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Volkov, V. V. & Svergun, D. I. Uniqueness ofab initioshape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003)

    CAS  Google Scholar 

  38. Svergun, D., Barberato, C. & Koch, M. H. J. CRYSOL - A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995)

    CAS  Google Scholar 

  39. Tria, G., Mertens, H. D. T., Kachala, M. & Svergun, D. I. Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCrJ 2, 207–217 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Bernadó, P., Mylonas, E., Petoukhov, M. V., Blackledge, M. & Svergun, D. I. Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem. Soc. 129, 5656–5664 (2007)

    PubMed  Google Scholar 

  41. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  PubMed  Google Scholar 

  42. Leslie, A. G. W. & Powell, H. R. Processing diffraction data with MOSFLM. Nato Sci Ser Ii Math 245, 41–51 (2007)

    Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  Google Scholar 

  45. 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 

  46. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    CAS  PubMed  Google Scholar 

  47. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)

    CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  49. Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods Mol. Biol. 1079, 263–271 (2014)

    PubMed  PubMed Central  Google Scholar 

  50. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    CAS  PubMed  Google Scholar 

  51. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nature Genet. 25, 25–29 (2000)

    CAS  PubMed  Google Scholar 

  54. Allen, C. L. & Gulick, A. M. Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein. Acta Crystallogr. D 70, 1718–1725 (2014)

    CAS  PubMed  Google Scholar 

  55. Lai, J. R., Fischbach, M. A., Liu, D. R. & Walsh, C. T. A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection. Proc. Natl Acad. Sci. USA 103, 5314–5319 (2006)

    ADS  CAS  PubMed  Google Scholar 

  56. 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 

  57. 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 

Download references

Acknowledgements

We thank T. Mintya, D. Avizonis, M.-C. Tang and A. Furtos for experimental support, R. Zamboni, J. Collucci and K. Guerard for small molecule synthesis assistance; J. Jiang, D. Alonzo and D. Rodinov for experimental advice and assistance; S. Labuik and P. Grochulski (Canadian Light Source) for diffraction data collection; R. Gillian (CHESS SAXS beamline G1), M. Pillon and A. Guarne for SAXS help: K. Bloudoff, M. Tarry, A. Haque and A. Beghuis for helpful discussions; and J. Pelletier, N. Rogerson and A. Nahvi for critical reading of the manuscript. This work was supported by CIHR grant 106615, a HFSP CDA and a Canada Research Chair in Macromolecular Machines to T.M.S. J.M.R. is supported by an NSERC Alexander Graham Bell studentship, and M.N.A. by a studentship from the CIHR Training Grant in Chemical Biology.

Author information

Author notes

  1. Janice M. Reimer and Martin N. Aloise: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, McGill University, 3649 Promenade Sir-William-Osler, Montréal, H3G 0B1, Québec, Canada

    Janice M. Reimer, Martin N. Aloise & T. Martin Schmeing

  2. Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montréal, H3A 1B1, Québec, Canada

    Paul M. Harrison

Authors
  1. Janice M. Reimer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin N. Aloise
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul M. Harrison
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. Martin Schmeing
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M.R. and M.N.A. cloned, expressed, purified, assayed, crystallized and determined the structure of LgrA F–A and F–A–PCP proteins. J.M.R. performed the CoA syntheses and prepared figures. P.M.H. performed the bioinformatics analyses. T.M.S. designed the study, analysed data and wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to T. Martin Schmeing.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Synthetic cycles in canonical initiation, canonical elongation and LgrA initiation modules.

Schematic diagrams comparing the synthetic cycle in canonical initiation and elongation modules with that in the LgrA initiation module.

Extended Data Figure 2 Representative electron density.

a–d, 2Fo − Fc density maps for protein in P41212 (a), R3:H (b), P21 (c) and P322 (d) crystal forms contoured at 1σ. e, f, Unbiased Fo–Fc density maps for the PPE–NH–Val arm in the P21 (thiolation state) contoured at 3.3σ (e), and a P41212 crystal soaked with N5–f THF, AMPcPP and valine contoured at 2.5σ (f).

Extended Data Figure 3 Crystal structures of the initiation module of linear gramicidin synthetase.

a–f, Models of F–A (Asub disordered) (a), F–A–PCP (PCP disordered) (b–d) and F–A–PCP from the four independent crystals structures determined (e, f). The crystal with space group P322 diffracted anisotropically to ~3.8 Å resolution, but the other higher resolution structures enabled the building of high quality models shown in d and f.

Extended Data Figure 4 Comparison between the LgrA initiation module and the SrfAC termination module.

a, b, The LgrA initiation module in the formylation state (a) and the termination module of surfactin synthase subunit 3 (SrfAC)22 (b) in the state where aminoacyl-PCP would be positioned to act as an acceptor substrate in the condensation reaction (PPE arm not present). The F and C domains are each positioned directly N-terminal of their A domains and bury similar amounts of A domain surface area (829 Å2 and 903 Å2; contributing residues shown in spheres), each forming ‘stable platforms’22. Both modules use very large movements of their PCP and Asub domains to bring the aminoacyl-PCP of the module to distant active sites to act as the acceptor substrate in an amide bond forming reaction. c, However, the F–A and C–A interfaces are distinct, and, if the A domains are superimposed, the F and C domains are only partially overlapping. This places their active sites in dissimilar locations, necessitating that Asub and PCP assume different positions to deliver their substrate. The PCP domain in the formylation state completely overlaps with the position of the C domain.

Extended Data Figure 5 Small-angle X-ray scattering analysis of F–A–PCP.

a, The crystal structure in the formylation state is shown superposed on the averaged filtered ab initio small-angle X-ray scattering model generated with DAMAVER37, with a NSD value of 0.819 ± 0.052. b, The calculated scattering curve for the DAMAVER is overlaid with the experimental scattering with χ2 = 3.010, where I represents scattering intensity and q is equivalent to 4πsin(θ)/λ. c, To understand the flexibility of F–A–PCP better, EOM39 was performed and generated five different ensembles. The ensemble resembling the formylation state structure represented over 60% of the optimized models generated, while the remaining <40% resembled the thiolation state structure. d, The calculated scattering of the EOM model has a χ2 = 1.028, which demonstrates that F–A–PCP has flexibility. The data are consistent with extreme flexibility for Asub and PCP domains, and limited flexibility in F–Acore. e, All independent molecules from the crystal structures were overlaid to further illustrate the flexibility of the system. f, CRYSOL38 was used to generate predicted scattering curves for the formylation state and thiolation state crystal structures with χ2 = 2.12 and χ2 = 5.54, respectively.

Source data

Extended Data Figure 6 Neighbour-joining tree of LgrA F domain and homologues.

This neighbour-joining tree of the LgrA F domain and homologues was made using PHYLIP (http://evolution.genetics.washington.edu) based on an initial Clustal Omega48 alignment of the closest 220 homologues of the LgrA F domain (Blast50 BLAST E-value <1 × 1014). The most similar formyltransferases to the F domain share ~45% identity, and all of these 220 formyltransferases have only inferred function. The tree was drawn using the program FigTree (http://tree.bio.ed.ac.uk). The sequences are named with their GenInfo Identifier (GI) numbers. Colouring: red, Brevibacilli; green, other Firmicutes; black, other bacteria; blue, Archaea. The clade of the LgrA F domain is highlighted in grey. Only nodes with bootstraps of >50% are shown. Several horizontal transfer events are evident where Firmicute and non-Firmicute proteins cluster together with high bootstrap values (for example, >70%). The several horizontal transfer events of formyltransferase domains between Firmicutes and other bacterial groups suggest the LgrA F domain likely originated from horizontal transfer.

Extended Data Figure 7 Neighbour-joining tree of LgrA A–PCP and homologues.

This neighbour-joining tree of LgrA A–PCP didomains and homologues was made for the 500 closest homologues (BLAST E-value <1 × 1014). The sequences are named with their GI accession codes. Colouring: red, Brevibacilli; green, other Firmicutes; black, other bacteria. The significant clades of the LgrA A–PCP domains are highlighted in grey. Only nodes with bootstraps of >50% are shown. Three functionally characterized homologues of LgrA that are shown to be directly related are labelled. The A–PCP portion of the initiation module is quite divergent, but the second module of LgrA clearly shares a common origin with functionally characterized NRPSs in Bacilli and other Firmicutes.

Extended Data Figure 8 Conservation and variation of residues involved in the interaction interfaces.

a, b, Sequence logos made using the WebLogo server (http://weblogo.berkeley.edu)52 show conservation and variation as found in multiple sequence alignments of F domain residues that interact with the A domain (a) and A domain residues that interact with the F domain (b). Below each logo are the corresponding residues in the LgrA proteins from the five Brevibacillus species, with the crystallized LgrA on the first line. FT, formyltransferase. c, d, Sequence logos indicate the conservation and variation in F domain residues involved in binding and interaction with PCP–PPE–Val across the closest 240 homologues of LgrA (c) and all of the functionally or structurally characterized formyltransferase proteins (d) (reduced for redundancy so that no two sequences have >50% sequence identity). e, Consensus sequences for the five Brevibacillus LgrA homologues and for the formyltransferases of known structure for each of three formyltransferase types. Catalytic residues are His73, Asn71 and Asp108.

Extended Data Figure 9 Interaction surfaces in PCP and Asub domains.

a, b, The Asub (a) and PCP2,25,54 (b) domains must maximize the use of their limited surfaces to interact with their many binding partners. Shown are the surfaces observed in this study, and many excellent previous studies have also documented interaction surfaces biochemically or structurally. This includes, for example, the equivalent of PCP domain residues Met249, Phe264 and Ala268, which are required for interaction with the C domain in the acceptor site55 and form hydrophobic interactions with the C domain22 in a very similar manner and using an overlapping surface, as the PCP domain does to interact with the F domain. Furthermore, partially overlapping surfaces in PCP domains have been proposed to interact with their (acyl-)PPE arm to protect thioester intermediates56 or to promote binding to the appropriate partner domain57. These interactions might occur during PCP domain transit, but they would have to be broken before productive binding to partner domains. Several of these PPE interactions are incompatible with the productive domain–domain interactions57, and in catalytic configurations seen here and previously, the PPE arms extend into the partner domain and make little contact with the PCP domain.

Extended Data Table 1 Crystallographic statistics
Full size table

Supplementary information

Supplementary Information

This file contains the legend for Supplementary Videos 1 and 2 with additional references. (PDF 148 kb)

Crystal structures of the initiation module of linear gramicidin synthetase

The structures of the initiation module of LgrA interpolated to visualize its entire synthetic cycle. The A domain is open for substrate binding (see also A domain structures2,20), and closes to catalyze valine adenylation formation (see also A domain structures2,18). Next, the Asub domain rotates 140° to catalyze transfer of the valine onto the thiol of the PPE arm of the PCP domain (see also A-PCP didomain structures19,21). Then the PCP domain transports its valine the 50 Å to the F domain to accept a formyl group. The PCP next moves the formyl-valine to the downstream elongation module, where it is passed off in that module’s condensation reaction (not shown), which liberates the PCP to participate in the next round of reactions. The experimentally observed structures are indicated by crystallographic statistics (disordered or absent portions for each structure coloured grey). For this animation, details of the adenylate in the A domain and the valine in formylation active site were based on co-complexes of isolated A domains58-62, A-PCP domains21,63,64 and FT proteins5,13,15,24. The extensive body of NRPS structural biology of protein constructs that contain A and/or PCP domains was also checked17,21,22,25,58-73 (see separate Supplementary Information file for additional references). (MOV 27353 kb)

Animation of the synthetic cycle of the initiation module of linear gramicidin synthetase

The structures of the initiation module of LgrA interpolated to visualize its entire synthetic cycle. The A domain is open for substrate binding (see also A domain structures2,20), and closes to catalyze valine adenylation formation (see also A domain structures2,18). Next, the Asub domain rotates 140° to catalyze transfer of the valine onto the thiol of the PPE arm of the PCP domain (see also A-PCP didomain structures19,21). Then the PCP domain transports its valine the 50 Å to the F domain to accept a formyl group. The PCP next moves the formyl-valine to the downstream elongation module, where it is passed off in that module’s condensation reaction (not shown), which liberates the PCP to participate in the next round of reactions. The experimentally observed structures are indicated by crystallographic statistics (disordered or absent portions for each structure coloured grey). For this animation, details of the adenylate in the A domain and the valine in formylation active site were based on co-complexes of isolated A domains58-62, A-PCP domains21,63,64 and FT proteins5,13,15,24. The extensive body of NRPS structural biology of protein constructs that contain A and/or PCP domains was also checked17,21,22,25,58-73. (see separate Supplementary Information file for additional references). (MOV 29669 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Reimer, J., Aloise, M., Harrison, P. et al. Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase. Nature 529, 239–242 (2016). https://doi.org/10.1038/nature16503

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature16503

This article is cited by

Search

Advanced search

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