8702
2007, 111, 8702-8704 Published on Web 07/04/2007
The Distribution and Conformation of Very Long-Chain Plant Wax Components in a Lipid Bilayer Eoin P. Coll,† Christian Kandt,† David A. Bird,‡ A. Lacey Samuels,‡ and D. Peter Tieleman*,† Department of Biological Sciences, UniVersity of Calgary, 2500 UniVersity DriVe NW, Calgary AB, T2N 1N4, Canada, and Department of Botany, UniVersity of British Columbia, VancouVer, Canada ReceiVed: June 1, 2007; In Final Form: June 14, 2007
Plant wax contains long-chain alkanes and related components which are transported to the surface of the plant by specialized ABC transporter proteins. Here, we determine the distribution and conformation of three wax components, nonacosane, nonacosan-15-one, and nonacosan-15-ol, using unbiased and umbrella sampling molecular dynamics simulations. The molecules all partitioned to the center of the bilayer, with a free-energy difference of -70 kJ/mol between bulk water and the center of the bilayer for the alkane and -55 kJ/mol for the two more-polar molecules. All of the wax molecules were highly mobile in the bilayer, freely moving between opposite leaflets on a time scale of a few nanoseconds. Nonacosan-15-one and nonacosan-15-ol folded double to expose their hydrophilic group to the solvent, whereas nonacosane alternated between orientations spanning the full bilayer and orientations in the center of the bilayer.
The aerial surface of all land plants is covered by a waxy cuticle composed of very long-chain fatty acids and their derivatives; for example, in the model plant Arabidopsis thaliana, the predominant cuticular “wax” components are C29H60 (nonacosane), 29-carbon ketones (C29H58O, specifically nonacosan-15-one), and 29-carbon secondary alcohols (C29H59OH, primarily nonacosan-15-ol).1 These protective lipids are synthesized in the epidermal cells of the plant and represent unusual secretory products due to their highly hydrophobic nature. The mechanism of wax synthesis and export is under examination in a series of Arabidopsis eceriferum (cer or glossy) mutants, in which the cuticular wax load is reduced due to mutations in genes encoding proteins required for either wax synthesis or export. Using these mutant lines, biosynthetic enzymes that produce the very long-chain fatty acid wax precursors, and some of their derivatives, have been identified and localized to the endoplasmic reticulum of epidermal cells. In addition, an ATPbinding cassette (ABC) transporter required for wax export was identified, using the cer5 mutant, which has reduced wax at the plant surface and accumulates intracellular lipidic inclusions.2 ABC transporters drive import or export by hydrolysis of ATP and are responsible for many important cellular functions.3 ABC transporters in humans are of interest as they are responsible for many genetic diseases as well as multidrug resistance in cancer chemotherapy.4 The structure and exact mechanism of operation of the CER5 ABC transporter have yet to be solved, although by homology comparison, it is a member of the ABCG/white-brown complex subfamily of ABC transporters, which includes lipid, sterol, and hydrophobic drug transporters.3 * To whom correspondence should be addressed. E-mail: tieleman@ ucalgary.ca. † University of Calgary. ‡ University of British Columbia.
10.1021/jp074265c CCC: $37.00
To understand both the biosynthesis and transport of wax components, the most energetically probable arrangement of these molecules in cells must be determined. This will inform studies of lipid-enzyme or lipid-transporter interactions and provide information as to which cellular compartments may contain the lipids. There are examples where a substrate, such as nonacosane, is modified by hydroxylation, but it is not known how the midchain alkane hydroxylase, the membrane-bound enzyme that adds the hydroxyl group to the center of the alkane chain, encounters its substrate. Similarly, how are wax components delivered to the CER5 ABC transporter? In this study, molecular dynamics (MD) simulations of wax components were used to characterize the partitioning of wax molecules in a dioleoylphosphatidylcholine (DOPC) bilayer, mimicking the plant membrane in which biosynthetic and transporter proteins reside. Unconstrained MD simulations and umbrella sampling simulations were performed using the Gromacs 3.3.1 suite.5,6 The OPLS force field with modifications by Berger et al.7 was used for all lipid and wax molecules, with water represented by the SPC model. The system comprised a membrane of 286 DOPC molecules with one nonacosane, nonacosone, or nonacosanol molecule and 10871, 9779, and 11520 waters, respectively, at a temperature of 298 K. The particle mesh Ewald8,9 method was used, with a Coulomb cutoff of 0.9 nm. Bond lengths were constrained with the LINCS10 algorithm; therefore, a 2 fs time step could be used. The system size was 9.5 × 9.5 × 8.0 nm, containing approximately 50000 atoms in all simulations. Umbrella sampling11 was used to obtain a potential of mean force (PMF) of the molecules throughout the membrane, as previously detailed in simulations of hexane in a lipid bilayer.12 The center of mass of each wax molecule was constrained at points 0.1 nm apart with a force constant of 3000 kJ mol-1 nm-2 in the direction normal to the bilayer. All other degrees © 2007 American Chemical Society
Letters of freedom remained unconstrained. Simulation was performed in 45 windows for 6 ns each. Two molecules of the same type were present in each system, offset by 4.5 nm, such that when one molecule was at the center of the bilayer, the other was in bulk water. This separation ensured that the molecules did not interact with one another yet produced two data sets for each simulation. Unconstrained 4 ns simulations contained only one wax molecule. Figure 1 shows the PMF obtained for each molecule. All of the wax components were highly hydrophobic, and a large positive free energy for removal of the waxes from the bilayer was expected and observed. Each molecule’s extended length of ca. 3.6 nm was sufficient to span almost the entire hydrophobic portion of the bilayer. In our umbrella sampling procedure, only the center of mass of the wax molecule was constrained; therefore, the molecules were free to adopt any rotational orientation. Figure 2 shows that the nonacosane most often adopts one of two orientations, either in the direction normal to the bilayer, aligned with the lipid tails, or extended in the plane of the bilayer in the low-density center. Evaluation of the cosines of the angles between the molecule’s axis and the direction normal to the bilayer showed that there is no discernible preferred orientation, and the molecule changed between the two orientations within several nanoseconds (details not shown). The slightly polar secondary nonacosan-14-ol and nonacosanone wax components have free-energy costs of approximately 59 kJ/mol for transfer from the center of the bilayer to bulk water. The nonacosane has a free-energy cost of removal from the bilayer of 75 kJ/mol. A previous simulation of hexane in a DOPC bilayer showed that partitioning of hexane to the center of the membrane was 24 kJ/mol more favorable than locating it in bulk water.12 Assuming that these hydrophobic molecules approximate spherical packing in water in order to minimize the exposed hydrophobic surface area, it is expected that free energy scales with surface area. As the nonacosane occupies 4.83 times the volume of hexane and has an associated surface area increase of 2.86 times, the expected free energy is 69 kJ/ mol. This value slightly underestimates the observed free energy of 75 kJ/mol. This is likely due to the fact that the molecules do not pack perfectly spherically in bulk water and that the nonacosane does not occupy the same space as the hexane molecule in the membrane, as it is longer and must occupy unfavorable areas near the water-membrane interface. An energy minimum for the secondary nonacosan-15-ol and nonacosanone was observed at 0.7-0.8 nm from the center of the bilayer. At this location, the hydrophobic regions of the waxes are oriented toward the center of the bilayer, and the polar ketone or hydroxyl groups are oriented toward the polar head region of the bilayer. A slight energy cost is associated with moving the molecules toward the center of the bilayer from this region, as the polar groups form hydrogen bonds at the 0.7-0.8 nm position, but no such interactions are possible at locations closer to the center of the bilayer. However, the nonacosanol has a second energy minimum at the center of the bilayer, possibly due to the lower density of this region reducing the entropic cost of locating a large molecule in the region. No such minimum was observed for the nonacosanone molecule, possibly due to the introduction of a kink into the molecule by the sp2-hybridized carbonyl group. These results show that wax components are effectively insoluble in water and favorably partition to the center of the cell membrane. Since more than half of the total fatty acids produced by an epidermal cell will be exported to the cell
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Figure 1. Free energy of three molecules in a DOPC bilayer. The black line represents nonacosane, the red line represents nonacosanone, and the green line represents nonacosanol. (A) The blue background represents the bulk water region, the red background represents the region of the polar head groups, and the yellow background represents the region of the lipid tails. (B) Individual potentials of mean force. As the PMF was expected to be symmetrical about the center of the bilayer, each PMF was mirrored about the zero position and averaged to obtain the potentials illustrated in A.
surface,13 this result has important implications for the properties of the biological membranes in these cells. Similar to more “conventional” export substrates such as carbohydrates and proteins, the waxes destined for the plant surface might be
8704 J. Phys. Chem. B, Vol. 111, No. 30, 2007
Letters in the membrane or binding can occur from a specific orientation, given the surprising flexibility of the substrate molecules. This may be of general relevance for ABC transporters and nonABC multidrug resistance proteins that take up hydrophobic molecules from the membrane. Acknowledgment. D.P.T. is an Alberta Heritage Fountain for Medical Research (AHFMR) Senior Scholar. C.K. is an AHFMR Postdoctoral Fellow. This work was supported by CIHR (D.P.T.) and NSERC (L.S.). References and Notes
Figure 2. Orientation of wax molecules in a DOPC bilayer. (A) A density plot of the alkane in bilayer over 4 ns of unconstrained simulation. The outer blue mesh depicts a wax density higher than 0.029 non-hydrogen atoms/Å3, showing the entire space sampled by the molecule. The inner red shape depicts a wax density of higher than 0.069 non-hydrogen atoms/Å3. (B) The lower energy conformation of the alcohol molecule in the DOPC bilayer. Grey spheres represent phosphate head groups, and blue spheres represent water oxygen atoms. The lipid tails have been omitted for clarity.
transported from the endoplasmatic reticulum to the cell membrane via Golgi-mediated secretion. It is unlikely that CER5 is a flippase, as the wax components readily flip from one leaflet of the cell membrane to the other on the order of a few nanoseconds, far faster than the rate of lipid exchange. If CER5 acts by taking up the wax components directly from the cell membrane and exporting them on the cell wall side, it either is capable of binding its substrates from a range of orientations
(1) Kunst, L.; Samuels, A. L. Prog. Lipid Res. 2003, 42, 51-80. (2) Pighin, J. A.; Zheng, H.; Balakshin, L. J.; Goodman, I. P.; Western, T. L.; Jetter, R.; Kunst, L.; Samuels, A. L. Science 2004, 306, 702-704. (3) Holland, I. B.; Cole, S. P. C.; Kuchler, K.; Higgins, C. F., ABC Proteins: From Bacteria to Man; Elsevier Science: New York, 2003. (4) Dean, M.; Rzhetsky, A.; Allikmets, R. Genome Res. 2001, 11, 1156-66. (5) Berendsen, H. J. C.; Spoel, D. v. d.; Drunen, R. v. Comput. Phys. Commun. 1995, 91, 43-56. (6) Lindahl, E.; Hess, B.; Spoel, D. v. d. J. Mol. Mod. 2001, 7, 306317. (7) Berger, O.; Edholm, O.; Jahnig, F. Biophys. J. 1997, 72, 20022013. (8) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 1008910092. (9) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577-8593. (10) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1984, 18, 1463-1472. (11) Torrie, G. M.; Valleau, J. P. J. Chem. Phys. 1977, 66, 1402-1408. (12) MacCallum, J. L.; Tieleman, D. P. J. Am. Chem. Soc. 2006, 128, 125-130. (13) Suh, M. C.; Samuels, A. L.; Jetter, R.; Kunst, L.; Pollard, M.; Ohlrogge, J.; Beisson, F. Plant Physiol. 2005, 139, 1649-1665.
