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J. Phys. Chem. B 2010, 114, 15712–15720
Structure, Dynamics, and Energetics of Lysobisphosphatidic Acid (LBPA) Isomers A. Goursot* and T. Mineva UMR 5253 CNRS/ENSCM/UM2/UM1 Ecole de Chimie de Montpellier, 8 rue de l′Ecole Normale, 34296, Montpellier, Cedex 5, France
C. Bissig and J. Gruenberg Department of Biochemistry, Sciences II, 30 quai Ernest Ansermet, 1211 GeneVa-4, Switzerland
D. R. Salahub Department of Chemistry and Institute for Biocomplexity and Informatics, UniVersity of Calgary, 2500 UniVersity DriVe NW, Calgary, Alberta, Canada T2N 1N4 ReceiVed: September 2, 2010; ReVised Manuscript ReceiVed: October 18, 2010
Lysobisphosphatidic acid (LBPA), or bis(monoacylglycerol)phosphate, is a very interesting lipid, that is mainly found in late endosomes. It has several intriguing characteristics, which differ from those of other animal glycerophospholipids, that may be related to its specific functions, particularly in the metabolism of cholesterol. Its phosphodiester group is bonded at the sn-1 (sn-1′) positions of the glycerols rather than at sn-3 (sn-3′); the position of the two fatty acid chains is still under debate but, increasingly, arguments favor the sn-2, sn-2′ position in the native molecule, whereas isolation procedures or acidic conditions lead to the thermodynamically more stable sn-3, sn-3′ structure. Because of these peculiar features, it can be expected that LBPA shape and interactions with membrane lipids and proteins are related to its structure at the molecular level. We applied quantum mechanical methods to study the structures and stabilities of the 2,2′ and 3,3′ LBPA isomers, using a step-by-step procedure from glycerol to precursors (in vitro syntheses) and to the final isoforms. The structures of the two positional LBPA isomers are substantially different, showing that the binding positions of the fatty acid chains on the glycerol backbone determine the shape of the LBPA molecule and thus, possibly, its functions. The 3,3′ LBPA structures obtained are more stable with respect to the 2,2′ form, as expected from experiment. If one argues that the in vivo synthesis starts from the present glycerol conformers and considering the most stable bis(glycero)phosphate structures, the 2,2′ isoform should be the most probable isomer. I. Introduction Lysobisphosphatidic acid (LBPA) or bis(monoacylglycero)phosphate is found in all animal tissues in a small amount (e1%) (Figure 1 where the acyl chain is oleoyl). However, in contrast to other phospholipids, LBPA is only detected in late endosomes where it accounts for ∼17 mol % of total membrane phospholipids.1,2 In addition, LBPA is abundant in the intralumenal vesicles that accumulate within late endosomes, accounting for the characteristic multivesicular appearance of these organelles.1 The function of lysophosphatidic acid in endocytic membranes is under active investigation. LBPA is involved in the traffic of proteins and lipids that transit via late endosomes.2,3 Late endosomal membranes rich in LBPA regulate cholesterol transport3 and assist in presenting sphingolipids to their degradation enzymes.4 Among LBPA isoforms, the 2,2′-dioleoyl isoform is the major component of the LBPA found in BHK cells (∼90 mol % of total LBPA). In a protein-free system, this 2,2′ isoform, but no other, spontaneously forms multivesicular liposomes that resemble the multivesicular endosomes where the lipid is found in vivo, provided that the liposome lumen is acidified to the endosome pH.5 This leads to the notion that LBPA may have the intrinsic capacity to deform membranes, leading to membrane invagination and vesicle formation. In addition, LBPA is highly resistant to lipases and phospholi* To whom correspondence should be addressed.
Figure 1. Schematic representation of LBPA with stereospecific numbering (sn) of the glycerol carbons.
pases,1,6-8 a property of the lipid that may account for its presence in the degradative environment of late endosomes. As a consequence, it has been proposed that the stereochemistry
10.1021/jp108361d 2010 American Chemical Society Published on Web 11/05/2010
Structure, Dynamics, and Energetics of LBPA Isomers of LBPA is unique: its stereochemical configuration would differ from that of other animal glycerophospholipids in that the phosphodiester moiety is linked to positions sn-1 and sn-1′ of glycerol, rather than to position sn-3 (Figure 1). Indeed, the in vivo synthesis of most glycerophospholipids involves the phosphorylation of glycerol at carbon 3, following the conventional stereospecific numbering (sn).9 It is interesting to note that pure dioleoyl-LBPA mono- or bilayers do not show any phase transition, whereas DPPC membranes containing LBPA are more fluid.10 It has been debated whether glycerol moieties were esterified with fatty acids in positions sn-3 or sn-2. However, recent studies indicate that the fatty acids are esterified at positions sn-2 and sn-2′ in the native molecule.5,11 Indirect information about the active LBPA isomer has been provided comparing the abundance of multivesicular vesicles formed at pH 5.5/7.4 with 2,2′- and 3,3′dioleoyl-LBPA, which concluded in favor of the former isomer. Moreover, 2,2′-dioleoyl LBPA, but not the 3,3′-isoform or other analogues, can restore cholesterol levels in cells from patients with the Nieman-Pick type C cholesterol storage disorder.12 Similarly, the cytosolic Alix protein was found to bind preferentially to liposomes containing 2,2′ LBPA.5 The mechanism of the in vivo LBPA synthesis is unknown and the molecular structure of this lipid has not been resolved. The observed deconstruction by LBPA of membrane bilayers has led to the thinking that the molecule, or a small aggregate of it, is distorted enough to perturb the bilayer organization. This effect of LBPA, as well as its proposed strong interaction with the ALIX protein, is thus pleading for theoretical investigations of its molecular structure. Moreover, we have recognized that the elucidation of lipid structures and, in particular, the recognition of possible conformers has to be based on quantum mechanical (QM) methods, which allow a quantitative evaluation of the molecular electronic energies.13 This paper explores the possible 2,2′ and 3,3′ LBPA isomeric structures and relative stabilities, using density functional theory (DFT). This exploration is based on earlier studies and debate on glycerolipid isomers. Whereas experimental NMR studies of phosphatidylcholine (PC) lipids at different concentrations in water proposed the presence and interchange of three possible rotational isomers around the glycerol C-C bond linked to the acyl chains,14 the intrinsic rigidity of the glycerol backbone has been invoked to interpret NMR dipolar coupling data for PCs.15 On the other hand, previous theoretical studies concluded that several isoenergetic isomers existed for PC lipids and showed that the glycerol molecule includes in its conformational space the precursors of the framework phospholipid conformers.13,16 Finally, recent in vitro synthesis protocols of the 2,2′ LPBA positional isomer have been reported, aimed at the unique acylation of the secondary glycerol carbons.11,17 The effect of the medium (pH, the nature of the counterion) on the acyl chain migration from 2,2′ to 3,3′ positions was underlined. In contrast, acidic late endosomes contain only the 2,2′ LBPA isoforms. This intriguing problem is tackled in this paper by using a novel computational strategy: computing and analyzing the geometries and electronic energies of LBPA precursors and LBPA isomers in terms of their glycerol and glycerophosphate building blocks. This step-by-step study led to successive interesting results. Whereas the 13C NMR glycerol data reveal the probable presence of only 2 preponderant isomers (A, B), the computed molecular stabilities of the LBPA precursors (in vitro syntheses) point toward one more favored bis(glycerol)phosphate structure based on the glycerol B isomer. The dynamical effects of water and temperature on these precursor models may enlarge the
J. Phys. Chem. B, Vol. 114, No. 47, 2010 15713 structural possibilities to rotating primary alcohol groups. Arriving at the LBPA molecular structure analysis, the most important result, probably not expected from a biological point of view, is that the binding positions of the acyl chains at the secondary and primary glycerol alcohols generate quite different structures for 2,2′ and 3,3′ low-energy isomers. It is thus tempting to speculate that the functions of LBPA in membrane deformation and transport are modulated by the composition of the acyl chain and by their position on the glycerol backbone. II. Methods All molecules have been studied with density functional theory (DFT) using a linear combination of atomic orbitals procedure, as implemented in the deMon2k program.18 All calculations were performed using the revised PBE exchange functional (revPBE)19 and the LYP20 correlation functional, augmented by a damped empirical correction for dispersionlike interactions,21 referred to as DFT-D. This DFT-D approach is necessary to account for the stabilizing interaction between the alkyl chains.22 DFT-optimized double-ζ plus valence polarization (DZVP) basis sets23 were employed for all atoms. For the fitting of the density, the GEN-A2 auxiliary function set was used.23 The exchange-correlation potential was numerically integrated on an adaptive grid.24 The evaluation of the relative stabilities of the glycerol isomers, as well as all chemical shielding calculations were performed using both the revPBE-LYP and PW9125 exchangecorrelation functionals, associated with DZVP and correlation consistent Dunning bases aug-cc-pVXZ (X ) D,T,Q).26 The auxiliary function set used in association with the augmented Dunning bases was enlarged to contain up to g functions (GENA2*).23 The grid accuracy was set to 10-5 in all calculations. The Coulomb energy was calculated by the variational fitting procedure proposed by Dunlap, Connolly, and Sabin.27,28 A quasi-Newton method in internal redundant coordinates with analytical energy gradients was used for the structure optimization.29 The convergence was based on the Cartesian gradient and displacement vectors with a threshold of 10-4 and 10-3 au, respectively. Born-Oppenheimer dynamics were performed for the most stable glycerol conformers and for synthetic precursors to LBPA. The temperature of the system was reached and maintained using the Nose´-Hoover chain thermostat30 in the NVT ensemble. The center of mass of the molecule was placed at the origin of coordinates and the linear and angular momenta were kept constant along the dynamics. The nuclear positions were updated using the velocity Verlet algorithm with a time step of 1 fs. The systems were equilibrated for 10 ps for glycerol and 15 ps for the model precursors in water (see below). The total equilibrated simulation time was 50 ps for the glycerol molecule in the gas phase and 20 ps for the LBPA precursors in water. III. Glycerol Conformers With its three hydroxyl groups, glycerol is highly soluble in water. In living organisms, it is the first precursor of triglycerides and phospholipids. Glycerol has been studied experimentally in its gas, liquid, and solid phases by infrared and Raman,31-35 electron and neutron diffraction spectroscopies,36-38 X-ray diffraction39 and by NMR as a neat liquid or in solution.38,40,41 Theoretical studies by quantum chemical31,42,43 and classical MD methods44,45 have been devoted to the conformational study of this molecule and to the assignment of the most probable
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J. Phys. Chem. B, Vol. 114, No. 47, 2010
Figure 2. Calculated structures of the six framework isomers of the glycerol molecule.
isomers present in the gas and liquid state. Figure 2 illustrates the six possible framework isomers of glycerol. There are many more minima on the molecular potential energy surface if one takes into account the OH rotations around the C-O bonds. In the gaseous state, more stable conformers correspond to a larger number of possible intramolecular O-H · · · OH hydrogen (H) bonds. As shown in Table 1, the most stable isomer found by all methods is indeed isomer F (or γγ in the nomenclature of ref 37) with three H bonds. A low-energy conformer with the same F backbone, called F′, has only two H bonds (the O-H on C1 is rotated by 120°). Isomers F′, A, and B with two H bonds have similar stabilities whereas isomers C, D, and E with only one H bond are less stable. As already reported,31,43 large basis set calculations lead to smaller energy differences between the most stable isomers F, F′, A, and B. Table 1 collects the results obtained with DFT (PW91), DFT-D (revPBE-LYP-D, see Section II), and MP2 methods and shows the effect of the basis set extension for the DFT results. The DFT results reveal that one needs the aug-cc-pVQZ basis to converge the electronic energy differences between the conformers (
