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. 2014 Jan 16;505(7483):427-31.
doi: 10.1038/nature12810. Epub 2013 Dec 22.

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

Chi Nguyen  1 , Robert W Haushalter  2 , D John Lee  2 , Phineus R L Markwick  3 , Joel Bruegger  4 , Grace Caldara-Festin  4 , Kara Finzel  5 , David R Jackson  4 , Fumihiro Ishikawa  5 , Bing O'Dowd  5 , J Andrew McCammon  6 , Stanley J Opella  5 , Shiou-Chuan Tsai  4 , Michael D Burkart  5
Affiliations

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

Chi Nguyen et al. Nature. .

Abstract

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. 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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Conflict of interest statement

Competing financial interests

The authors have none.

Figures

Figure 1
Figure 1. E. coli AcpP and crosslinking strategy
a, AcpP is a small, acidic protein comprised of four α-helices that interacts with at least 19 catalytic enzymes, 12 of which belong to FAS (10 shown here). The apolar interior of helix II (α2) and helix III (α3) form a hydrophobic cavity that sequesters the growing metabolite attached to the PPant arm. b, (top) A native substrate of FabA and (middle) modified AcpP with targeted sulfonyl-3-alkynyl crosslinking probe, derived from (bottom) the crosslinking pantetheinamide analog 1. c, Proposed mechanism of FabA. Protein-protein interactions between AcpP and FabA induce release of the sequestered substrate from AcpP into the active site of FabA, where dehydration is catalyzed. d, Crosslinking strategy to form AcpP=FabA with mechanism-based crosslinking probe 1.
Figure 2
Figure 2. Structure of crosslinked AcpP=FabA
a, X-ray crystal structure of AcpP=FabA at 1.9 Å. b, The molecular surface mapped with calculated vacuum electrostatic potential of AcpP=FabA. Blue shading indicates electro-positive and red shading indicates electro-negative protein surfaces. c, Rotating b 90° at the interfaces between each AcpP=FabA to visualize electrostatic pairing. d, Expanded view of both interfaces in AcpP=FabA, indicating salt bridges and hydrophobic interactions between helix II (α2) and helix III (α3) of AcpP and the Positive Patch of FabA. e, Comparison between hydrophobic cleft of AcpP with (top) sequestered substrate (from PDB: 2FAE, with long interior hydrophobic cavity outlined with dashed line) and (bottom) AcpP1 in AcpP=FabA (reduced interior cavity). f, The interior cavity of 2FAE labeled with the hydrophobic residues. The contraction of these hydrophobic residues collapses the interior cavity in AcpP=FabA.
Figure 3
Figure 3. NMR studies
a, HSQC spectra of 15N-octanoyl-AcpP in the absence of FabA (green), and with increasing (yellow to red) concentrations of FabA. Chemical shift perturbations (CSPs) are observed in AcpP residues that interact with FabA or the bound acyl chain. In magenta is the overlaid HSQC of 15N-AcpP=FabA. b, Expanded views of select residues. c, CSPs were measured for each 15N-octanoyl-AcpP residue in the absence and presence of 1 molar equivalent of FabA and plotted by residue number. d, AcpP residues from c. where CSPs larger than 0.065 ppm are indicated in red. e, CSPs measured between 15N-octanoyl-AcpP and the 15N-AcpP=FabA were measured and plotted by residue number. f, AcpP residues from e. where CSPs larger than 0.25 ppm are indicated in red. In NMR convention, protein residue number precedes residue letter; the converse applies with crystallography.
Figure 4
Figure 4. Molecular dynamics and protein-protein interactions
a, Experimental RDC data correlated with theoretical RDCs. (left) 15N-octanoyl-AcpP (black) with Pf1 bacteriophage and (red) 5% neutral charge compressed polyacrylamide gel, and (right, magenta) crosslinked AcpP=FabA with Pf1 bacteriophage. b, Order parameter calculations of (left) octanoyl-AcpP and (right) AcpP=FabA. Nanosecond (blue) timescale compared to microsecond [RDC-optimized] (dotted) timescale routines. c, Sausage plot of order parameter differences on the microsecond timescale between octanoyl-AcpP and AcpP=FabA. Color and thickness depict relative disorder, where red represents maximal difference of 0.5. (Detailed in SI.) d, Residues of the Positive Patch mediating protein-protein interactions in known structures. (blue) FabA, (cyan) COL (PDB: 4DXE), (purple) stearoyl ACP desaturase (PDB: 2XZ0), (orange) ACPS (PDB: 1F80), (magenta), ACP-P450 (PDB: 3EJB), (yellow) ACP-STAS (PDB: 3NY7), and (green) ACP-BioH (PDB: 4ETW).
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