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Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

Abstract

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis1. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain2. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway3,4,5. The transient nature of ACP–enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein–protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP–FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4′-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

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Figure 1: E. coli AcpP and crosslinking strategy.
Figure 2: Structure of crosslinked AcpP–FabA.
Figure 3: NMR studies.
Figure 4: Molecular dynamics and protein–protein interactions.

Accession codes

Accessions

Protein Data Bank

Data deposits

The atomic coordinates of AcpP–FabA have been deposited in the Protein Data Bank under accession 4KEH.

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Acknowledgements

M.D.B. and S.-C.T. are supported by GM100305 and GM095970. We thank J. J. LaClair for figure editing. We thank X. Huang for assistance with NMR facilities and experimental setup. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Advanced Light Source is supported by the Office of Basic Energy Sciences of the US Department of Energy under contract no. DE-AC02-05CH11231. J.A.M. is supported by NSF, NIH and HHMI. Portions of the research are supported by the Advanced Light Source, supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231.

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Contributions

C.N., assisted by G.C.-F., D.R.J. and J.B., determined the AcpP–FabA X-ray crystal structures. R.W.H., D.J.L. and B.O. conducted the protein NMR experiments under the supervision of S.J.O. F.I. and K.F. prepared the crosslinking probe. P.R.L.M. conducted molecular dynamics simulations under the supervision of J.A.M. C.N., G.C.-F., R.W.H. and D.J.L. analysed data and contributed to writing of the paper. S.-C.T. and M.D.B. directed the research, provided funding and wrote the final manuscript.

Corresponding authors

Correspondence to Shiou-Chuan Tsai or Michael D. Burkart.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-18, Supplementary Methods, Supplementary Tables 1-9, a Supplementary Discussion and additional references. (PDF 15899 kb)

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Nguyen, C., Haushalter, R., Lee, D. et al. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis. Nature 505, 427–431 (2014). https://doi.org/10.1038/nature12810

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