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

Nature volume 505, pages 422–426 (2014)Cite this article

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Abstract

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.

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Accession codes

Accessions

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.

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Acknowledgements

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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Author notes

  1. Christian R. H. Raetz: Deceased.

Authors and Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Ali Masoudi, Christian R. H. Raetz & Pei Zhou

  2. Human Vaccine Institute, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Charles W. Pemble IV

  3. Duke Macromolecular Crystallography Center, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Charles W. Pemble IV

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  1. Ali Masoudi
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Contributions

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.

Corresponding author

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). https://doi.org/10.1038/nature12679

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