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Chasing acyl carrier protein through a catalytic cycle of lipid A production


Acyl carrier protein represents one of the most highly conserved proteins across all domains of life and is nature’s way of transporting hydrocarbon chains in vivo. Notably, type II acyl carrier proteins serve as a crucial interaction hub in primary cellular metabolism1 by communicating transiently between partner enzymes of the numerous biosynthetic pathways2,3. However, the highly transient nature of such interactions and the inherent conformational mobility of acyl carrier protein2 have stymied previous attempts to visualize structurally acyl carrier protein tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but also of therapeutic value. For example, acyl carrier protein is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides in Gram-negative microorganisms, which is required for their growth and survival4,5, and is an activator of the mammalian host’s immune system6,7, thus emerging as an important therapeutic target8,9,10. During lipid A synthesis (Raetz pathway), acyl carrier protein shuttles acyl intermediates linked to its prosthetic 4′-phosphopantetheine group2 among four acyltransferases, including LpxD11. Here we report the crystal structures of three forms of Escherichia coli acyl carrier protein engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures show the intricate interactions at the interface that optimally position acyl carrier protein for acyl delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled acyl carrier proteins provide the molecular basis for the association–dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber shows an unprecedented role of acyl carrier protein in product release.

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Figure 1: Stalled ACPs bound to LpxD.
Figure 2: Intermolecular interactions between ACP and LpxD.
Figure 3: ACP conformations and reorganization of its prosthetic group.
Figure 4: Molecular basis for the ordered-sequential reaction mechanism and involvement of ACP in lipid-product release.

Accession codes


Protein Data Bank

Data deposits

Molecular coordinates and structure factors of intact-acyl-ACP, hydrolysed-acyl-ACP and holo-ACP in complex with LpxD have been deposited in the Protein Data Bank under accession numbers 4IHF, 4IHG and 4IHH, respectively.


  1. Butland, G. et al. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433, 531–537 (2005)

    Article  CAS  ADS  Google Scholar 

  2. Byers, D. M. & Gong, H. Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649–662 (2007)

    Article  CAS  Google Scholar 

  3. White, S. W., Zheng, J., Zhang, Y. M. & Rock The structural biology of type II fatty acid biosynthesis. Annu. Rev. Biochem. 74, 791–831 (2005)

    Article  CAS  Google Scholar 

  4. Galloway, S. M. & Raetz, C. R. H. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. Biol. Chem. 265, 6394–6402 (1990)

    CAS  PubMed  Google Scholar 

  5. Belunis, C. J., Clementz, T., Carty, S. M. & Raetz, C. R. H. Inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli. J. Biol. Chem. 270, 27646–27652 (1995)

    Article  CAS  Google Scholar 

  6. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998)

    Article  CAS  ADS  Google Scholar 

  7. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. Cell 124, 783–801 (2006)

    Article  CAS  Google Scholar 

  8. Onishi, H. R. et al. Antibacterial agents that inhibit lipid A biosynthesis. Science 274, 980–982 (1996)

    Article  CAS  ADS  Google Scholar 

  9. Williams, A. H., Immormino, R. M., Gewirth, D. T. & Raetz, C. R. H. Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide. Proc. Natl Acad. Sci. USA 103, 10877–10882 (2006)

    Article  CAS  ADS  Google Scholar 

  10. Jenkins, R. J. & Dotson, G. D. Dual targeting antibacterial peptide inhibitor of early lipid A biosynthesis. ACS Chem. Biol. 7, 1170–1177 (2012)

    Article  CAS  Google Scholar 

  11. Raetz, C. R. H., Reynolds, C. M., Trent, M. S. & Bishop, R. E. Lipid A modification systems in Gram-negative bacteria. Annu. Rev. Biochem. 76, 295–329 (2007)

    Article  CAS  Google Scholar 

  12. Bartling, C. M. & Raetz, C. R. H. Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis. Biochemistry 47, 5290–5302 (2008)

    Article  CAS  Google Scholar 

  13. 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  CAS  ADS  Google Scholar 

  14. Nooren, I. M. & Thornton, J. M. Diversity of protein-protein interactions. EMBO J. 22, 3486–3492 (2003)

    Article  CAS  Google Scholar 

  15. Chan, D. I. & Vogel, H. J. Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem. J. 430, 1–19 (2010)

    Article  CAS  Google Scholar 

  16. Ploskon, E. et al. Recognition of intermediate functionality by acyl carrier protein over a complete cycle of fatty acid biosynthesis. Chem. Biol. 17, 776–785 (2010)

    Article  CAS  Google Scholar 

  17. Roujeinikova, A. et al. Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates. J. Mol. Biol. 365, 135–145 (2007)

    Article  CAS  Google Scholar 

  18. Buetow, L., Smith, T. K., Dawson, A., Fyffe, S. & Hunter, W. N. Structure and reactivity of LpxD, the N-acyltransferase of lipid A biosynthesis. Proc. Natl Acad. Sci. USA 104, 4321–4326 (2007)

    Article  CAS  ADS  Google Scholar 

  19. Bartling, C. M. & Raetz, C. R. H. Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis. Biochemistry 48, 8672–8683 (2009)

    Article  CAS  Google Scholar 

  20. Raetz, C. R. H. & Roderick, S. L. A left-handed parallel β helix in the structure of UDP-N-acetylglucosamine acyltransferase. Science 270, 997–1000 (1995)

    Article  CAS  ADS  Google Scholar 

  21. Frederick, A. F., Kay, L. E. & Prestegard, J. H. Location of divalent ion sites in acyl carrier protein using relaxation perturbed 2D NMR. FEBS Lett. 238, 43–48 (1988)

    Article  CAS  Google Scholar 

  22. Cryle, M. J. & Schlichting, I. Structural insights from a P450 carrier protein complex reveal how specificity is achieved in the P450(BioI) ACP complex. Proc. Natl Acad. Sci. USA 105, 15696–15701 (2008)

    Article  CAS  ADS  Google Scholar 

  23. Parris, K. D. et al. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8, 883–895 (2000)

    Article  CAS  Google Scholar 

  24. Kraut, D. A., Carroll, K. S. & Herschlag, D. Challenges in enzyme mechanism and energetics. Annu. Rev. Biochem. 72, 517–571 (2003)

    Article  CAS  Google Scholar 

  25. Kelly, T. M., Stachula, S. A., Raetz, C. R. & Anderson, M. S. The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis. J. Biol. Chem. 268, 19866–19874 (1993)

    CAS  PubMed  Google Scholar 

  26. Agarwal, V., Lin, S., Lukk, T., Nair, S. K. & Cronan, J. E. Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis. Proc. Natl Acad. Sci. USA 109, 17406–17411 (2012)

    Article  CAS  ADS  Google Scholar 

  27. Babu, M. et al. Structure of a SLC26 anion transporter STAS domain in complex with acyl carrier protein: implications for E. coli YchM in fatty acid metabolism. Structure 18, 1450–1462 (2010)

    Article  CAS  Google Scholar 

  28. Holak, T. A., Nilges, M., Prestegard, J. H., Gronenborn, A. M. & Clore, G. M. Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry. Eur. J. Biochem. 175, 9–15 (1988)

    Article  CAS  Google Scholar 

  29. Roujeinikova, A. et al. X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site. Structure 10, 825–835 (2002)

    Article  CAS  Google Scholar 

  30. Jiang, Y., Chan, C. H. & Cronan, J. E. The soluble acyl-acyl carrier protein synthetase of Vibrio harveyi B392 is a member of the medium chain acyl-CoA synthetase family. Biochemistry 45, 10008–10019 (2006)

    Article  CAS  Google Scholar 

  31. Rock, C. O., Cronan, J. E., Jr & Armitage, I. M. Molecular properties of acyl carrier protein derivatives. J. Biol. Chem. 256, 2669–2674 (1981)

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Qiu, X. & Janson, C. A. Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions. Acta Crystallogr. D 60, 1545–1554 (2004)

    Article  Google Scholar 

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

    Article  Google Scholar 

  37. Schuttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004)

    Article  Google Scholar 

  38. 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 

  39. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  Google Scholar 

  40. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  41. DeLano, W. L. The PyMOL Molecular Graphics System v. 1.3r1 (Schrödinger, LLC, New York, New York, 2012)

  42. Stokes, G. B. & Stumpf, P. K. Fat metabolism in higher plants. The nonenzymatic acylation of dithiothreitol by acyl coenzyme A. Arch. Biochem. Biophys. 162, 638–648 (1974)

    Article  CAS  Google Scholar 

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We acknowledge our co-author Christian R. H. Raetz, who shaped the lipid field with his curiosity and efforts, introducing many scientists to the field during his renowned career. We thank R. Brennan and W. Todd Lowther for reviewing the manuscript. Finally, we thank Z. Guan for the help with the mass spectrometry of ACP, H.-S. Chung and other members of Raetz laboratory, as well as J. M. Burg, for discussions. Crystal screening, data collection and data processing were conducted in collaboration with the Duke Macromolecular X-ray Crystallography Shared Resource. Diffraction data were collected remotely at the Southeast Regional Collaborative Access Team 22-BM and 22-ID beamlines at the Advanced Photon Source, Argonne National Laboratory, supported by the US Department of Energy, Office of Science and the Office of Basic Energy Sciences under Contract number W-31-109-Eng-38. This work was supported by National Institutes of Health grants GM-51310 and AI-055588 awarded to C.R.H.R. and P.Z.

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Authors and Affiliations



A.M., C.R.H.R. and C.W.P. designed research; A.M. performed all biochemical experiments under the guidance of C.R.H.R., P.Z. and C.W.P.; A.M. performed all protein expression, purification and crystallization; A.M. and C.W.P. contributed to data collection, structure solution and refinement; A.M., C.R.H.R. and C.W.P. analysed and interpreted the structures; A.M. and C.W.P. made the figures and wrote the manuscript; A.M., C.R.H.R., P.Z. and C.W.P. discussed the results and commented on the manuscript.

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Correspondence to Charles W. Pemble IV.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2 and Supplementary Figures 1-13. Additional details about the cloning primers and sequences, crystallographic data statistics, and structural analysis for the ACP-LpxD complexes are given. Also shown are detailed sequence comparisons with orthologs and biochemical evidence for acyl-ACP hydrolysis and the role of ACP in the LpxD reaction mechanism. (PDF 9957 kb)

Supplementary Data

This file contains the Source Data for Supplementary Figure 12. (XLSX 65 kb)

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Masoudi, A., Raetz, C., Zhou, P. et al. Chasing acyl carrier protein through a catalytic cycle of lipid A production. Nature 505, 422–426 (2014).

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