Solvation and Hydration of the Ceramide Headgroup in a Non-Polar

Article pubs.acs.org/JPCB

Solvation and Hydration of the Ceramide Headgroup in a Non-Polar Solution Richard J. Gillams,† Jon V. Busto,‡ Sebastian Busch,† Félix M. Goñi,‡ Christian D. Lorenz,*,§ and Sylvia E. McLain*,† †

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K. Unidad de Biofísica (CSIC, UPV-EHU) and Departamento de Bioquímica, Universidad del País Vasco, P.O. Box 644, 48080 Bilbao, Spain § Department of Physics, King’s College London, London WC2R 2LS, U.K. ‡

S Supporting Information *

ABSTRACT: The microscopic hydration of the ceramide headgroup has been determined using a combination of experimentalboth NMR and neutron diffraction techniques and computational techniquesempirical potential structure refinement (EPSR) and molecular dynamics (MD). The addition of water to ceramide in chloroform solutions disrupts the chloroform solvation of the ceramide headgroup, and the water forms distinct pockets of density. Specifically, water is observed to preferentially hydrate the two hydroxyl groups and the carbonyl oxygen over the amide NH motif. Further assessment of the location and orientation of the water molecules bound to the ceramide headgroup makes it clear that the strongly solvated carbonyl moiety of the amide bond creates an anchor from which water molecules can bridge via hydrogen bonding interactions to the hydroxyl groups. Moreover, a significant difference in the hydration of the two hydroxyl groups indicates that water molecules are associated with the headgroup in such a way that they bridge between the carbonyl motif and the nearest neighbor hydroxyl group.

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INTRODUCTION

ceramides contain a fatty acyl chain of 16 carbon atoms or more. Both the acyl tails2 and the sphingoid base3 may influence their membrane properties. In general, they are among the least polar, more hydrophobic lipids in membranes.4 Indeed, their hydrophobicity explains their abundance in the stratum corneum, the barrier that prevents water evaporation through the skin.5 Their solubility in water is negligible; thus, free ceramides cannot exist in solution in biological fluids or in the cytosol. Ceramides exhibit a large range of biological effects that can vary, sometimes dramatically, with small changes in molecular structure. Ceramides were related to programmed cell death, or apoptosis;6,7 they have been later associated with several processes of stress signaling8 including some instances of human disease.9 Among the unusual physical properties of ceramides in membranes,10,11 their tendency to associate with each other has attracted the attention of different investigations12−16 (see reviews in refs 10 and 11). Ceramides are not homogeneously dispersed in the membrane bilayer but rather give rise to laterally separated micron-sized domains, containing up to 40 mol % ceramide.17 This is considered to occur as a result of a rich network of H-bonds linking the headgroups of neighboring ceramides.11,17,18 The capacity of the ceramide headgroup

Ceramides (N-acyl sphingosines) (Figure 1) constitute the hydrophobic backbones of all the complex sphingolipids, i.e., sphingomyelin, cerebrosides, and gangliosides,1 but they exist free in membranes as well. The most commonly found

Received: October 27, 2014 Revised: December 12, 2014 Published: December 12, 2014

Figure 1. Chemical structure of ceramide, highlighting the labels used to describe the different atoms throughout this text. © 2014 American Chemical Society

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DOI: 10.1021/jp5107789 J. Phys. Chem. B 2015, 119, 128−139

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The Journal of Physical Chemistry B

structure for a number of biomolecules in solution has been determined.21,24−27 Further details of EPSR are presented elsewhere.23,28 In order to ensure a reliable model of the structure, a range of samples are measured, which are chemically equivalent but contain a variety of well-defined isotopic substitutions. The substituted atomsin this case hydrogen and deuteriumhave different scattering lengths bα (eq 1), such that each sample produces a distinct scattering pattern. Integration of the RDFs, extracted from the EPSR model, gives rise to a local coordination number (nβα(r)), which describes the average number of β atoms around a central α atom within a distance range from rmin to rmax via

(Figure 1) to exchange H-bonds was noted from the early physical studies of this molecule;19,20 however, a detailed study of this property is still lacking. A basic step in understanding the ceramide H-bonding network is a detailed study of ceramide headgroup solvation, of the kind performed previously for phosphocholine,21 the polar moiety of important phospholipids, e.g., phosphatidylcholine and sphingomyelin. The aim of the current study is to gain an understanding of the solvation of the ceramide headgroup in solution. Specifically, this investigation focuses on the interactions between water and the ceramide headgroup in water/ chloroform solutions, where water has been titrated into the ceramide chloroform solutions in order to gain an understanding of the hydration of this very hydrophobic lipid in solution. This has been achieved through a combination of experimental and computational approaches in order to gain the greatest level of understanding of the system at an atomic/ molecular level.

nαβ = 4πρcβ

∑

(2 − δαβ)cαcβbαbβ Sαβ(Q ) (1)

where δαβ is the Kronecker delta function and Sαβ(Q) are the partial structure factors for each atom pair. Partial structure factors are directly related to the distances in real spacethe radial distribution functions (RDFs), gαβ(r)via Fourier transformation by Sαβ(Q ) = 1 + 4πρ

∫ r 2[gαβ(r) − 1] sin(QrQr) dr

r 2g (r ) dr

(3)

In the current work, a series of samples were measured in which ceramide was dissolved in different isotopic mixtures of chloroform, specifically with deuteration levels of 0, 25, 50, 75, and 100%. The molecular ratios for each sample were 1:158 ceramide:chloroform, heretoafter referred to as Cer-0. Neutron diffraction data were collected on the samples using the SANDALS instrument at the ISIS Facility (STFC, UK) at 45 °C. Each of the samples were contained in canisters constructed from a Ti/Zr alloy, which have a negligible scattering length and measured for ∼1500 μA each. Similar measurements were obtained from the empty sample cans as well the empty instrument and a vanadium standard in order to ensure adequate background subtraction and data normalization. The neutron data were corrected for absorption, multiple scattering, and inelastic background contributions using the program Gudrun, which is based on the ATLAS package29 and is available at the ISIS neutron source. In addition, analogous samples to those used for the NDIS were prepared for characterization via SANS and measurements were recorded on the LOQ instrument in order to determine whether there was any large scale aggregation within the samples (these are shown in the Supporting Information). X-ray Diffraction. A sample was prepared for X-ray diffraction (XRD) at the same concentration as the 0% deuterated NDIS sample described above and loaded into a borosilicate glass capillary with a diameter of 1 mm. The sample was measured on a Panalytical X’pert Pro X-ray diffractometer at ISIS (UK), which has a Q range of 0.7−18 Å−1 at 45 °C, and the data were corrected using GudrunX. Empirical Potential Structure Refinement. The EPSR simulation box contained 32 ceramide molecules and 5056 chloroform molecules at the temperature and density of the measurements (T = 318 K; ρ = 0.03937 atoms Å−3). The seed potentials used for the atoms, bonds, and bond angles for the ceramide molecules were taken from a CHARMM based model,30 the chloroform potentials from OPLS. These seed potentials are shown in the Supporting Information along with the experimental data and the respective EPSR fits to the data. Molecular Dynamics. Simulations were performed using the CHARMM force field.31 A model for ceramide has recently been proposed for the CHARMM force field,30 and this was combined with a chloroform model originally parametrized for the OPLS/AA force field.32 The water was modeled using TIP3P water33 modified for use with CHARMM parameters.31 Two systems were simulated, the first with 32 ceramide molecules dissolved in 5056 chloroform molecules (Cer-0), the same molecular ratio as the measured data and EPSR

MATERIALS AND METHODS Sample Preparation. Ceramide was purchased from Avanti Polar Lipids (Alabama, USA) and used without further purification. CHCl3 and CDCl3 were purchased from SigmaAldrich (U.K.) and were dried over either CaH2 or P2O5 and stored over 4 Å molecular sieves under a N2 atmosphere prior to use. While chloroform and water are not miscible in large quantities, a small amount of water can be solubilized in chloroform.22 For this reason, it was important to ensure that the chloroform used for sample preparation was adequately dried. For the neutron diffraction and NMR measurements, ceramide in chloroform both with and without water was prepared by weight. The samples were heated and shaken to ensure dissolution of the ceramide into the chloroform. Neutron Diffraction with Isotopic Substitution. Neutron diffraction enhanced by isotopic substitution (NDIS) provides access to the structure factor of a sample, F(Q), in reciprocal space. The structure factor is a function of the concentration, c, and scattering length, b, of the different atoms within the sample and can be described as α ,β≥α

rmax

min

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F (Q ) =

∫r

(2)

where ρ is the density of the sample given in units of atoms Å−3 and gαβ(r) is the RDF for β atoms around α atoms over a range of distances, r, from the central atom. In complex solutions, with many components, extracting the individual partial structure factors for the individual atoms in the system requires a computational model. In the current work, empirical potential structural refinement (EPSR, described below), a simulation technique which fits the experimentally observed diffraction patterns and has been designed specifically to model amorphous systems,23 is used to provide a model of the structure. The combination of neutron scattering with EPSR is a well established method by which the 129


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Figure 2. 1H NMR spectra of ceramide in chloroform (red) and upon the addition of 0.6 water molecules (Cer-0.6; blue).

snapshots of the trajectory. The analysis presented here is averaged across all ceramide molecules (32 for Cer-0 and 33 for Cer-0.6) and over all 5000 frames of the trajectory. As a result, the plots shown describe the solvation of ∼15 000 ceramide snapshots. The output from this analysis is then converted into a three-dimensional map using SDM mapping code written in Python,41 using the scipy42 and matplotlib43 packages, to produce three-dimensional plots with Mayavi.44 The SDMs depict the region of the central molecule used for the analysis, with isocontour “clouds” of density surrounding it. The cloud coverage can be tuned in order to determine the percentage of molecules which are surrounded by the isocontour shapesby increasing the percentage of molecules, the density at which the surface is drawn is decreased and so a larger area is included inside the shape.

simulations. The second system contained 33 ceramide molecules and 19 water molecules in 5214 molecules of chloroform, which is a concentration of 0.6 waters per ceramide molecule (Cer-0.6). These systems were run for 100 ns during the production stage of the simulations using the LAMMPS molecular dynamics code.34 In preparation for the production run, the systems were initially minimized with a conjugate gradient, followed by equilibration runs in both NVE and NVT ensembles. The electrostatic and van der Waals forces were calculated using a switching function, to reduce the interactions to zero between the cutoffs of 10 and 12 Å, with long-range interactions calculated using the particle−particle/particle− mesh K-space algorithm. The temperature and pressure were maintained using a Nosé−Hoover thermostat and barostat at 318 K and 1 atm, respectively.35,36 The pressure was coupled isotropically due to the liquid nature of the system. All bonds involving hydrogen atoms were treated with the SHAKE algorithm,37 enabling the use of a 2 fs time step. NMR. A sample was prepared for NMR containing ceramide in fully deuterated chloroform for the Cer-0 system at the same concentration as was measured for the diffraction experiments. 1 H NMR spectra were recorded, as well as 1H−13C-HSQC, COSY, TOCSY, and NOESY spectra on an in-house 600 MHz instrument. Subsequent to measuring ceramide in anhydrous chloroform, 0.6 waters per ceramide were added to the sample under a nitrogen flow (Cer-0.6) and the same spectra were recorded as for Cer-0. ANGULA. Spatial density maps (SDMs) were produced, which show the three-dimensional location of the molecules relative to one another, using the program ANGULA.38−40 Within ANGULA, coordinate axes can be assigned to different parts of the molecules in the system which can be used to calculate the distances, angles, and Euler angles between the coordinate systems, enabling a detailed picture of the location and orientation of molecules relative to one another. As this analysis focuses on a very localized area, different parts of a molecule can have different coordinate system assignments; for example, in the ceramide headgroup, three regions are analyzed independently. The use of axes to describe the position of the central molecule for these calculations allows ANGULA to consider the location of the surrounding atoms relative to a specific region of a molecule. As a result, these relative positions can be gathered from different ceramide molecules within a single snapshot of the simulation as well as from different

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RESULTS AND DISCUSSION Proton Environments in Ceramide. The 1H NMR spectra for ceramide in anhydrous CDCl3 (Cer-0) and for hydrated ceramide (Cer-0.6) in the same solvent are shown in Figure 2. The peak assignments correspond to the labeling shown in Figure 1 (the unlabeled hydrogen atoms carry the same numbering as the carbons that they are connected to) and are also listed in tabular form in the Supporting Information. The spectra here show good agreement with previous NMR investigations for ceramide in more dilute chloroform solutions45,46 with the exception of the OH peak shifts which appear at 2.85 ppm in Figure 2. However, in the previous investigations, it was estimated that 2 waters per ceramide were present by integration of the hydroxyl peak area45 where the presence of these water molecules will affect the hydroxyl chemical shift. The absence of water in the anhydrous Cer-0 spectrum recorded here (Figure 2; red spectra) is confirmed by the lack of an obvious water peak, which is observed in bulk chloroform at ∼1.50 ppm (relative to the CHCl3 at 7.26 ppm) at 45 °C (see the Supporting Information), and integration of the OH peaks gives a value of ∼2 protons. In addition, in Figure 2, the H(O)1/H(O)2 peaks at ∼2.8 ppm show splitting, but as the chemical shifts for both H(O)1 and H(O)2 are almost identical, the exact nature of the coupling cannot be determined readily. The assignment of the individual OH peaks was further verified via 2D COSY and TOCSY spectra (shown in the Supporting Information). 130


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Figure 3. NOESY NMR spectra of ceramide in chloroform (red/orange) and upon the addition of 0.6 water molecules for every ceramide molecule (blue/purple).

As water is added to the ceramide−chloroform system, a number of changes in the spectra in Figure 2 are observed. The presence of water is confirmed by a peak which appears at ∼1.7 ppm upon the addition of 0.6 waters per ceramide (Figure 2;

blue line). The hydroxyl group peaks become indistinguishable and appear as a singlet which is shifted downfield to ∼2.9 ppm. The amide group Hn proton similarly experiences a downfield shift, although the effect is not as pronounced as for the 131


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Figure 4. RDFs showing the distribution of chloroform-H (Hc) around the ceramide headgroup in both the Cer-0 and Cer-0.6 systems. Left-hand side, Cer-0; right-hand side, Cer-0.6. Top, around carbonyl (O); middle, first hydroxyl (O1); bottom, second hydroxyl (O2). The RDFs calculated from EPSR are shown as blue dashed lines, with the MD calculations shown in red (Cer-0) and green (Cer-0.6) lines.

the ceramide headgroup for both Cer-0 and Cer-0.6. Overall, in Figure 4, the MD and EPSR for the Cer-0 system show good agreement with one another. The chloroform hydrogen (Hc) solvates the carbonyl moiety in preference to the hydroxyl groups, as evidenced by the prominent first peak present at ∼3 Å in the Hc-O function. While this peak is also present in the hydroxyl-Hc RDFs, it is not as prominent, which suggests that the chloroform hydrogen is less likely to solvate either hydroxyl oxygen compared with the CO oxygen in the ceramide headgroup. This is also evident when calculating the average coordination (via eq 3), which shows that the chloroform hydrogen has a greater affinity for the carbonyl oxygen (n = 1.4) than for O1 and O2, which have coordination numbers of 1.1 and 0.9, respectively (see Table 1). It is interesting to note that the solvation of the two hydroxyl groups is not identical, which may be linked to the proximity of the hydroxyl groups to the amide group.

hydroxyl groups. Interestingly, the water resonance in the Cer0.6 spectrum is at 1.7 ppm as opposed to 1.5 ppm where water appears in pure chloroform at a similar temperature, which may be reflective of water being tightly bound to the ceramide headgroup as has been previously suggested.45,47 NOESY spectra were recorded for both Cer-0 and Cer-0.6 systems and are shown in Figure 3. Similar to the spectra in Figure 2, the majority of the peaks are unaffected by the presence of water with the exception of the H(O)1 and H(O)2 peaks which merge. The water resonance displays cross-peaks with the downfield-shifted −OH peaks, indicating that water is located close to the hydroxyl protons. The comparison of these spectra emphasizes the absence of water in the Cer-0 system, as there is no visible peak for water. The intensity of the crosspeak for the interaction of the Hn peak in the Cer-0.6 spectra is significantly lower than that of the hydroxyl protons. This indicates that the association of water with the amide NH group is less significant, thus suggesting a preference for hydration of the hydroxyl groups. This finding is consistent with previous analyses of crystallographic studies on the hydration of proteins where CO2− groups were found to be more hydrophilic than corresponding amide groups.48 Chloroform Solvation Structure. Radial distribution functions (RDFs), which describe the distribution of atoms relative to one another, were extracted from both the MD trajectories (Cer-0 and Cer-0.6) and the EPSR fits to the diffraction data (Cer-0). Figure 4 shows the RDFs for the chloroform hydrogen (Hc) atoms around the polar regions of

Table 1. Intermolecular Coordination Numbers for Chloroform-H around the Ceramide Headgroup, Measured at 4 Åthe Minimum after the Sharp Peak in the RDFs in Figure 4

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central molecule

Cer-0 EPSR

Cer-0 MD

Cer-0.6 MD

carbonyl O first hydroxyl O1 second hydroxyl O2

1.41 1.08 0.88

1.44 1.25 0.81

1.38 1.22 0.81


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Figure 5. SDMs showing the distribution of chloroform carbon around the ceramide headgroup from the MD simulation in the Cer-0 system (a−c) compared with the Cer-0.6 system (d−f). 6% of the density is shown for the carbonyl oxygen (O) solvation (a and d), the O1 (b and e), and O2 (c and f).

forming a band around the carbonyl oxygen. The OH oxygen atoms in the Cer-0 system on the other hand are preferentially solvated by a band of molecules in front of the zy plane in the +x-direction, where this band is extended in the +z-direction for the O2 hydroxyl oxygen (Figure 5c and f). Upon the addition of water molecules to the system, there is a slight decrease in chloroform solvation around the CO oxygen, which is also observed in the coordination number for the Hc-O RDF in Table 1. This subtle decrease in solvation can be seen through the change in color of the isocontour surfaces between Figure 5a and d. The chloroform solvation of the hydroxyl groupsO1 and O2remains largely unaffected upon the addition of water in the Cer-0.6 simulation, which is consistent with the smaller change in coordination number seen in Table 1. Water Interactions with Ceramide. Figure 6 shows the RDFs for the hydration of the ceramide headgroup. From this figure, it is clear that water is more likely to form hydrogen bonds with the carbonyl oxygen, as the first peak in the RDFs for the gOHw(r) function is considerably more prominent compared to the analogous RDFs for water−hydroxyl oxygen interactions. This is confirmed by the coordination numbers for these interactions which are found in Table 2. It should be noted that inspection of the MD trajectory indicated that the water−ceramide hydrogen bonds are highly fluctional, as during the course of the simulation water molecules freely moved between ceramide headgroups (see the Supporting Information). The SDMs for these interactions, shown in Figure 7, illustrate the locations where the water replaces some of the chloroform solvation around the oxygen sites in the ceramide headgroup when viewed in conjunction with Figure 5. Consistent with the solvation RDFs, a comparison of the water density distributions in the SDMs show that the carbonyl

The chloroform solvation of ceramide in the Cer-0.6 system, shown on the right-hand side of Figure 4, is comparable to that seen in the Cer-0 system. Similarly, the average coordination (shown in Table 1) is similar between the solutions with a very slight decrease in coordination observed for O and O1 atoms upon the addition of water. In order to understand this solvation in three dimensions, spatial density maps (SDMs) were generated from the MD trajectories using ANGULA (as described in the Materials and Methods section). These are shown in Figure 5. The SDMs are comprised of three parts. First, the chemical structure of the part of the ceramide for which the axis system is defined is shown in the center. The second aspect is a cross section of the solvent density, which is shown on the panels surrounding the SDMs. These do not show a projection of the density but rather a cross section through the center of the axis system, which has then been displaced by a distance of 6 Å. The third component is a density cloud, which is shown as an isocontour surface, surrounding the most densely solvated regions in threedimensional space. These SDMs were produced by mapping all chloroform molecules within 8 Å of the functional group onto the appropriate coordinate system and using an isocontour surface to enclose 6% of the chloroform carbon (Cc) atoms with the highest probability density. The side bar shows the color scale used to show the density of the solvent molecules. The density is on an absolute scale, and the same color scale applies to the isosurface and the cross sections on the panels in Figure 5. From this figure, it can be seen that the carbonyl oxygen (O; Figure 1) site is solvated by chloroform molecules in a distribution around the oxygen in the +z-direction (Figure 5a and d). This chloroform (CO) solvation pattern is reminiscent of the hydration of carbonyl oxygen atoms within other biological molecules in solution,49,50 with the density 133


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Figure 6. Radial distribution functions showing the distribution of water around the ceramide headgroup. Top, around amide group; middle, around first hydroxyl; bottom, around second hydroxyl.

Table 2. Intermolecular Coordination Numbers n for WaterH and Water-O around the Ceramide Headgroup, Measured at 2.5 Å central molecule

nHw

nOw

carbonyl O first hydroxyl O1/H(O)1 second hydroxyl O2/H(O)2 nitrogen amide N/Hn

0.10 0.03 0.01 0.00

0.01 0.01 0.01

distribution of chloroform shown in Figure 5a, with most of the water hydration being directly above the CO oxygen in the +z direction. In particular, there is a greater density of water molecules around the carbonyl on the side closest to the O1 hydroxyl oxygen. This can be seen by the position of the isocontour shape and, perhaps more clearly, through the red region in the cross section of the density profile which is seen on the panel (in the xz plane) of the plot. This appears to be a highly directed effect in comparison to the ring of density seen for the chloroform distribution and highlights a significant difference in solvation from what would be expected for a solvent-accessible carbonyl group.26,50 The difference in hydration of the hydroxyl groups is also more

group is most densely solvated by both chloroform and water. However, the SDM of water around the carbonyl (O in Figure 7a) shows a skewed distribution of water compared with the

Figure 7. SDMs showing the distribution of water oxygen around the ceramide headgroup from the MD simulation in the Cer-0.6 system. These plots show the top 14% of the most densely populated regions for the hydration of ceramide in the Cer-0.6 MD simulation. 134


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the reference atom, while an angle of 0° indicates that the water is oriented such that the vector bisecting the two hydrogen atoms of the water molecule is pointing directly at the reference atom. Figure 8 shows the distribution of ϕ1 angles for the three different oxygen locations in the headgroup. The carbonyl oxygen, O, shown as black bars, gives a typical distribution of angles for hydrogen bonding to an isolated hydrogen bond acceptor, with a distribution of angles centered around ∼50°. This is consistent with the hydrogen atoms being directed toward the carbonyl oxygen, such that the O−Hw−Ow is linear. This very directed orientation suggests that the water is bound quite strongly to the carbonyl group and does not rotate freely. However, the distribution of angles around the two hydroxyl groups shows significant distortion away from this arrangement, especially in the case of the second hydroxyl group, O2. This effect can be rationalized through two competing interactions. First, the carbonyl can only accept hydrogen bonds, while the hydroxyl groups can both donate and accept hydrogen bonds. This means that there is a second orientation where the Ow is accepting a hydrogen bond from the H(O)1/H(O)2 as well as the O1/O2 accepting hydrogen bonds from the Hw. This would account for the secondary peak seen at approximately 120°. The second aspect which affects the distribution of the ϕ1 angles around the hydroxyl groups is the influence of the carbonyl moiety. Due to the preferential solvation of the carbonyl group seen above, many of the waters solvating the hydroxyl groups will also be interacting with the carbonyl. As a result, the orientation of the water molecules around the hydroxyl group is not just determined by the influence of the hydroxyl groups but is instead a combination of the influence of both the hydroxyl and carbonyl groups, such that these waters are restricted in the way in which they can approach the hydroxyl groups. Conformation of Ceramide. In order to determine whether the hydration of ceramide is linked to a conformational change in the headgroup, the intramolecular RDFs were compared for the Cer-0 and Cer-0.6 systems. These are shown in Figure 9, and the associated coordination numbers are shown in Table 3. From this figure, H(O)1 protons show a much higher probability of being coordinated at a distance of around 2 Å with either O or O1 atoms, compared with the H(O)2 protons, which show much smaller peaks for both O and O1 associated RDFs at this distance range. A comparison of coordination numbers emphasizes this higher H(O)1oxygen correlation, where roughly half of the H(O)1 atoms are bound to either O or O1 in both MD and EPSR fits to the diffraction data, as opposed to 10−20% of H(O)2oxygen contacts at this distance. This is unaffected by the addition of water for Cer0.6, where, in the presence of water, the carbonyl group is still more frequently bound to the first hydroxyl group, O1 and H(O)1. The interaction with the second hydroxyl group, O2 and H(O)2, is much less probable, as the coordination number for this peak is relatively low (Table 3). For the amide nitrogen, a large, sharp peak for both Cer-0 and Cer-0.6 in the MD simulations indicates that the CO···H(O)1 and O2···H(O)1 conformations are invariant with respect to the amide nitrogen both with and without the presence of water molecules. The MD and EPSR are broadly similar, except with respect to the NH(O)1 function where there is a broader distribution of amideH(O)1 distances seen in the EPSR fits. This difference is probably due to the fact that in MD molecules are held more

marked. The O2 containing OH group (Figure 5c) is hydrated directly above the hydrogen (in the +z-direction), while O1 (Figure 5b) is also hydrated above the OH hydrogen but also shows density in front of the yz-plane. When comparing the hydration of both of these OH groups in the +z-direction, there is a higher density of nearest water neighbors of O1 compared with O2. Viewed as a whole, the combination of the hydration SDMs for O and O1 suggests that water molecules may bridge the gap between the amide and this OH within the ceramide headgroup in this predominantly hydrophobic environment. In addition to the location, the orientation of the water molecules has also been assessed. In order to do this, the angles at which water interacts with different regions of the ceramide headgroup were recorded from the MD simulations. The angles were subsequently divided into 5° bins, from which a histogram was generated. Vectors were defined within the water molecule and between the water molecules and the ceramide oxygen atoms. The first vector is defined in the direction from the water oxygen, Ow, to the reference atom (e.g., the carbonyl oxygen, O). The second vector is defined such that it bisects the Hw−Ow−Hw angle (from Ow to a point halfway between the two Hw atoms, shown schematically in Figure 8). The angle formed between these two vectors is labeled ϕ1. By defining the second vector to bisect the Hw−Ow−Hw angle, both Hw atoms feature at an angle of ∼52°, making it simpler to consider both Hw atoms. For example, an angle of 180° indicates that the water molecule is oriented such that its oxygen is nearest

Figure 8. Distribution of water orientations found within the MD simulation of the Cer-0.6 system. The angle ϕ1 is defined be the vectors V⃗ 1 (between Ow and the reference atom (for example, O1)) and V⃗ 2, which bisects the water molecule, as shown schematically at the top of this figure. The plot shows the distribution of ϕ1 angles, around the three areas where most hydration is seen: the carbonyl oxygen (black), the first hydroxyl oxygen (red), and the second hydroxyl oxygen (blue). 135


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Figure 9. Intramolecular radial distribution functions showing the distribution of polar regions of the ceramide headgroup around one another labeled as shown in Figure 1.

molecules are highly mobile. Interestingly, while water clearly interacts with the headgroup and often with multiple parts of the headgroup, it does not appear to bring about a conformational change in the headgroup itself. A representative structure from the MD trajectory which depicts this headgroup hydration is shown in Figure 10. This structural motif may be

Table 3. Intramolecular Coordination Numbers for the Different Polar Parts of the Ceramide Headgroup around One Another, Measured at 2.8 Å interaction

Cer-0 EPSR

Cer-0 MD

Cer-0.6 MD

O−H(O)1 O−H(O)2 O2−H(O)1 O1−H(O)2 N1−H(O)1 N1−H(O)2

0.24 0.07 0.22 0.13 0.11 0.04

0.36 0.02 0.23 0.09 0.58 0.09

0.33 0.02 0.25 0.10 0.55 0.08

rigid than they are in EPSR where a more disordered distribution of molecules is more likely. The similarity between the Cer-0 and Cer-0.6 systems, in terms of both RDFs and coordination numbers, indicates that there is no dramatic conformational change in the ceramide headgroup upon the addition of water.

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CONCLUSIONS From this investigation, there appears to be a tendency for both chloroform and water to solvate ceramide preferentially around the carbonyl oxygen (O, Figure 1). The conformation adopted by the ceramide headgroup is dictated by a significant amount of intramolecular hydrogen bonding and the introduction of water to the system leads to regions of hydration which subtly change these intramolecular interactions, with water bound to the headgroup. From visual inspection of the MD trajectory, the water molecules do not appear to be strongly bound to any particular part of the headgroup but rather show that the water

Figure 10. Snapshot from the MD simulation of the Cer-0.6 simulation, which captures a water molecule bound to both the carbonyl of the amide group and the hydroxyl group, bridging across the headgroup.

due to a combination of the transient nature of the water interactions and the intramolecular hydrogen bonds that dictate the conformation of the headgroup in the Cer-0 system. It is clear that the first hydroxyl group is much more frequently involved in hydrogen bonding than the other polar sites within the ceramide headgroup, either to its neighboring 136


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gents.1,14 The lack of associated water may be a requirement for the reduction of the lamellar to gel phase transition temperature.1 In the present paper, it would appear that the affinity for ceramide to interact with itself is more marked than its affinity for water. This can be seen by comparison of intermolecular coordination numbers for water interacting with ceramide in comparison to the intramolecular coordination numbers for ceramide. This might be due to the low concentration of water in the system, but it could also imply that the ceramide head groups have a preference to be dehydrated and close to one another, rather than separated by water. In a phospholipid membrane, the penetration of water into the interfacial region leads to a significant contribution to the overall pressure in this part of the membrane.51 However, in the case of ceramide, the low affinity for water would allow much tighter packing and thus increase the propensity for the ceramide head groups to pack more closely together.11 Ceramide is implicated in hemolysis in erythrocytes,52 through the breakdown of sphingomyelin by the PlcHR2 enzyme. The presence of ceramide in the form of ceramideenriched rigid domains introduces a susceptibility to hot−cold hemolysis. The introduction of ceramidase, which digests the ceramide to sphingosine, leads to cell leakage, but the cells are then resistant to hot−cold hemolysis. The formation of ceramide-rich domains has also been implicated in the activity of membrane-bound enzymes, such as phospholipase A2.14 When ceramide is a gel state, the area per lipid has been reported at 37.8,47 40,53 and 42 Å2.54 Simulations of ceramide have reported that, when the gel is melted, there is a significant increase in area per lipid from ∼42 to ∼54 Å2.30 This would imply that, if ordered domains were to form in a cell membrane aided by the self-association of ceramide molecules, a driving force would be provided to reduce the surface area. The contents of the cell do not change without rupturing the membrane, so a decrease in surface area could lead to a buildup in pressure and increased stress on the membrane. It is possible that this change could be absorbed by a reduction in undulations in the membrane surface and may be insignificant if there is not a very high concentration of ceramide leading to very large areas of domain formation. However, the tight intramolecular binding seen in the ceramide headgroup observed here may be a contributing factor leading to cell leakage and cell lysis. Ceramide clearly plays a diverse range of roles in different environments, depending upon many factors, such as the other components of the lipid bilayer and the medium surrounding the bilayer. Ceramide molecules in the skin stratum corneum are in a very different environment from those on erythrocytes or other cells. Within the skin, the barrier properties which ceramide can enhance are crucial. This is reliant on the lipids adopting a very rigid gel phase. In this environment, it has been shown that the ability to repel water even in the case of pores within the bilayer is also critical.55 The affinity of ceramide to form intramolecular hydrogen bonds in preference to binding water may help explain why ceramide is particularly effective in this role. In summary, the ceramide headgroup hydration observed here is relevant in the explanation of important ceramide properties, namely, their high hydrophobicity, their tendency to form laterally separated domains (i.e., their low miscibility with phospholipids in membranes), and their capacity to form nonlamellar (inverted hexagonal) structures

amide group, or with a water molecule, where this binding motif may be indicative of a water bridging interaction from the O1/H(O)1 to the amide group. This is somewhat surprising, as the number of chemical bonds between the amide and both of the hydroxyl groups is identical. The main difference in the local chemical structure between the two hydroxyl environments is the tail region, which is closer to H(O)2, where the proximity of this acyl tail may hinder the mobility of this hydroxyl group, thus limiting its interactions with the amide. In turn, this may be related to the fact that it is the O1/H(O)1 hydroxyl group, i.e., the C1 hydroxy, and not O2/H(O)2, that is enzymatically phosphorylated as the prelude for further metabolic modifications of ceramide. The addition of water disrupts the chloroform solvation of the ceramide headgroup, and water forms distinct pockets of density around the headgroup. The NMR experiments show that the amide group is less well hydrated compared with the hydroxyl groups, and this relative hydration is also consistent with both the EPSR and MD simulations. However, the NMR experiments cannot provide definitive information about the hydration of the O0 (due to the lack of an associated proton), or a comparison between H(O)1 and H(O)2, as the hydroxyl peaks merge upon the addition of water to the solution, restricting the information available. The details of hydration were observed via both MD and EPSR fits to the diffraction data, allowing further exploration of the aspects inaccessible by NMR, highlighting the importance of combining these techniques which give a consistent picture of ceramide hydration in these largely hydrophobic solutions. The work presented here considers ceramide molecules in solution rather than in a micelle or bilayer, where the molecules are free to adopt a wide range of conformations. Within a biological membrane, there is more water present on the outside of the membrane than in the solutions measured here, and the lipid tails will be constrained to the interior of the membrane. In addition to the confinement of the tails, the ceramide headgroup itself is fairly rigid, as it contains an amide group and an unsaturated C−C bond, and further rigidity will be conferred by the hydrogen bonds, which will limit the possible conformations of the headgroup in a membrane. Within both the EPSR fits to the diffraction data and the MD simulations, the ceramide molecules adopt both an extended conformation (with the tails extending in opposing directions from the headgroup) and a hairpin conformation (with the tails lying together in a fashion which would be comparable with the membrane environment). Both of these conformations can adopt either intramolecular hydrogen bonds or electrostatic contacts which are at longer distances yet in contact by virtue of bridging water molecules. It is interesting to observe that there appears to be no water tightly bound to the ceramide molecules. This is evident in the hydration coordination numbers for the RDFs where the highest water coordination to ceramide for the CO oxygen suggests that, on average, only 10% of water molecules are bound to this group (see Table 2). Moreover, summing over the entire headgroup, only around 15% of the water molecules in the simulation box are bound to ceramide on average. This indicates that water is easily displaced from the headgroup in solution and, perhaps, by extension in a biological membrane. In addition, the lack of tightly bound waters may be critical for allowing ceramides to pack very tightly with other lipids and cholesterol in membranes, leading to the formation of domains with reduced mobility and increased resistance to deter137


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which in turn can give rise to cell membrane invagination and fission.56

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ASSOCIATED CONTENT

S Supporting Information *

Additional tables and figures showing seed potentials for EPSR simulations, comparison of the MD and EPSR with the NDIS data, additional NMR details, and small angle neutron scattering data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Christina Redfield (Oxford) for help with the NMR measurements and Luis Carlos Pardo (Barcelona) for help with ANGULA analysis. We would also like to acknowledge the use of the IRIDIS High Performance Computing Facility provided by the Science & Engineering South (SES) Centre for Innovation. S.E.M. wishes to thank the UK Engineering and Physical Sciences Research Council for funding (EP/J002615/ 1) and the ISIS Facility (STFC, UK) for the allocation of neutron beam time. F.M.G. (Bilbao) thanks the Spanish Ministerio de Economia for grant BFU 2012-36241. J.V.B. is a postdoctoral scientist supported by the Basque Government.

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REFERENCES

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Solvation and Hydration of the Ceramide Headgroup in a Non-Polar

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