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GD1a in Phospholipid Bilayer: A Molecular DynamicsSimulationDebjani Roy a & Chaitali Mukhopadhyay aa Department of Chemistry , University of Calcutta , 92, A.P.C. Road, Calcutta , 700 009 ,IndiaPublished online: 15 May 2012.

To cite this article: Debjani Roy & Chaitali Mukhopadhyay (2001) GD1a in Phospholipid Bilayer: A Molecular DynamicsSimulation, Journal of Biomolecular Structure and Dynamics, 18:4, 639-646, DOI: 10.1080/07391102.2001.10506695

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GD1a in Phospholipid Bilayer: A Molecular Dynamics Simulation

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Abstract

Molecular dynamics simulation of ganglioside GD1a attached to the upper layer of a fullyhydrated lipid bilayer of dimyristoyl phosphatidyl choline (DMPC) at room temperatureunder periodic boundary conditions was performed. The time average conformation ofGD1a reveals that the terminal sialic acid is more exposed into the solvent than the internalbranched one. Many interresidual contacts between N-acetyl galactosamine-internalbranched sialic acid; external Gal-external sialic acid; N-acetyl galactosamine-internal galare also observed. The conformation of the GD1-hexasaccharide is stabilized by a numberof intra molecular hydrogen bonds that were previously observed experimentally. The sim-ulation results indicate that the presence of a single GD1a molecule has local effects on thebilayer. A local disorder in the arrangement of the acyl chains as well as the head groups isevident in the upper layer due to the presence of GD1a.

Introduction

Gangliosides are a group of complex glycosphingolipids (GSLs) characterized bythe presence of one or more acidic sugar units known as sialic acid (1). They consistof an oligosaccharide part, which is attached to a lipid known as ceramide.Oligosaccharide part contains a neutral backbone consisting of two to four neutralsugar units [for example, asialo-GM1: Gal β(1-3) GalNAc β(1-4) Gal β(1-4) Glcβ]and the 3- position of the inner galactose is always glycosylated with one sialic acidunit. When more than one sialic acids are present, they are attached to the 3-positionof the outer galactose. The ganglioside GD1a [NeuAc α(2-3) Gal β(1-3) GalNAcβ(1-4) {NeuAc α(2-3)}Gal β(1-4) Glcβ-O-ceramide (Figure 1) contain two sialicacid residues and they are attached to both the inner and the external galactose unitsof the neutral tetrasaccharide core (asialo-GM1). The gangliosides are distributed pri-marily on the plasma membranes of most vertebrates and are especially enriched inbrain and nervous tissues. The carbohydrate chains of the gangliosides that protrudefrom the cell surface, play a key role in cell-cell adhesion, recognition, differentiationand signal transduction (2-7) and as receptors for toxins (8-11). Gangliosides areknown to undergo specific structural changes during oncogenic transformations (12).

In order to understand the biological function of these molecules, a knowledge ofthe three-dimensional structure is essential. So far there is no single crystal X-raydiffraction data available on GD1a. However NMR techniques have been widelyapplied in structural and conformational studies on GD1a (13-16). Scarsdale et al.(1990) (13) suggested that a prominent source of stabilization for the native GD1astructure is the presence of a rather extensive hydrogen-bonding network betweenthe two sialic acid residues. It has been shown by Sabesan et al.(1991) (14) that inthe lowest energy conformer of GD1aOS (only hexasaccharide fragment) the inter-nal, branched sialic acid is stacked underneath the GalNAc residue, establishing ahydrogen bond between the carboxylate group of the sialic acid and the acetamido

Journal of Biomolecular Structure &Dynamics, ISSN 0739-1102Volume 18, Issue Number 4, (2001)©Adenine Press (2001)

Debjani Roy and Chaitali Mukhopadhyay*Department of Chemistry,

University of Calcutta,

92, A.P.C. Road,

Calcutta - 700 009, India

639

*Phone: xx-xx-xxx-xxxx; Fax: 91-33-351-9755; E-mail: chaitali@cucc.ernet.in

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group of the GalNAc unit. Both sialic acid residues are oriented almost perpendi-cular to the plane of the neutral trisaccharide core. The three dimensional structuresof the gangliosides GalNAc-GD1a and GD1a have been modeled using molecularmechanics and dynamics calculations, based on NOE interactions observed fornative gangliosides dissolved in deuterated dimethylsulfoxide or as mixed micelleswith fully deuterated dodecylphosphocholine in D20 by Acquotti et al.(15). It hasbeen found that compared with GD1a the GalNAc-GD1a is less mobile, thus influ-encing the surface area. It has been shown that the conformation of the trisaccha-ride core GalNAc-(Neu5Ac-)Gal in dimethylsulfoxide solution is well definedbecause many interresidue contacts between the residues. The external sialic acidhas been found to behave differently from the inner one. The four different con-formers of GD1a has been reported.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1aanchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution,were probed by high resolution NMR spectroscopy (Poppe et al., 1994) (16) Theobserved 1H/1H NOE interactions revealed conformational averaging of the terminalNeuAc α(2-3) Gal and Galβ(1-3) GalNAc glycosidic linkages. GD1a gangliosidecontaining an acetyl group as the acyl moiety, GD1a(acetyl), was studied in aqueoussolution by Brocca et al. (1995) (17) by laser light-scattering measurements.GD1a(acetyl) spontaneously aggregates as small micelles showing a hydrodynamicradius and molecular mass of 33Å and 96 kDa, respectively. Vibrio cholerae sialidaseshowed a very high activity on the micelles of GD1a(acetyl), compared to GD1a.

In the above reports GD1a was studied either as a free monomer (GD1os) whereceramide residue was released by mild base treatment or as inserted in a micelle ofphospholipid in aqueous solution. A characterization at the molecular level of theassociation of GD1a with membranes which simulates a multilamellar lipid bilay-er and not as isolated monolayer is necessary for a better understanding of its dif-ferent important biological activities. Molecular dynamics simulations represent apowerful approach to understand such complex systems. In recent years it has beenused to gain insight into the structure and dynamics of pure lipid membranes (18-21) as well as their interaction with small solutes (22) and proteins (23-27). In thepresent paper we report studies on MD simulation of ganglioside GD1a, anchoredin a model dodecylphosphocholine bilayer with inclusion of explicit solvent.

Methods

All molecular mechanics and dynamics calculations reported here were performedusing the program DISCOVER [with the Consistent Valence Force Field (CVFF)](28,29) as implemented in InsightII molecular modeling package of MSI Inc. Thisforcefield has recently been modified to include carbohydrate atom types (30). It hasalso been used successfully to model N-linked oligosaccharides and other gangliosidesand has been proven to be adequate (31-33). Acquotti et al. (1994) (15) have used bothCVFF and AMBER* (i.e. AMBER modified for carbohydrates) for the study of GD1aand found that two force fields gave similar values for all the glycosidic linkages, thedifferences being less than 19° except for ϕ of Galβ(1-4)Glc linkage (30°) and bothfound the similar lowest energy minimum (Acquotti et al. 1994) (15).

GD1a hexasaccharide was made by adding one sialic acid to the terminal galactose ofthe GM1 pentasaccharide obtained from X-ray crystallographic studies of choleratoxin- GM1 complex by Merritt et al. (1998) (34) (PDB code 3CHB). The structure ofGD1a hexasaccharide is shown as Figure 1. Sphingosine and stearic acid chains ofGD1a were built by modeling using BIOPOLYMER module in InsightII.Subsequently these were attached to the GD1a hexasaccharide. The resulting GD1amolecule was employed for energy minimization until the rms deviation of energy wasless than 0.001 kcal/mol. The minimized structure of the GD1a was then subjected todynamics simulation at room temperature (300°K) for 100 ps initial equilibration fol-

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lowed by a 500 ps simulation run. The time step of the dynamics simulation was 1 fs.

The initial conformation of DMPC molecules were built by modeling using BIOPOLY-MER module in InsightII. The DMPC molecule thus generated was then minimised for1000 steps using conjugate gradient method. This energy minimized conformation wasthen employed to dynamics simulation in vacuum at room temperature (300°K) for250 ps. 16 DMPC molecules were randomly chosen from this dynamics trajectory (0.5ps interval) and were arranged into a membrane bilayer with 8 DMPC molecules ineach layer. The fifth DMPC molecule from the upper layer was removed. The GD1amolecule was introduced in the void created by removal of one DMPC molecule fromthe bilayer of 8 DMPC molecule with the sugar containing part facing away from thecore of the membrane. Figure 2 shows the starting conformation of bilayer with GD1aattached to it. As a control a DMPC bilayer without GD1a was also constructed. Boththe systems (bilayer+GD1a and bilayer only) were extended with periodic images in xand y dimensions. The third dimension (z) also extended so that the GD1a images sur-rounded by the lipids form continuous two dimensional membrane slabs separated bylayers of water. The x, y and z dimensions of the bilayer were 49Å, 55Å and 16Årespectively. Awater box of 539 molecules of water has been added to the system. Onesystem thus consists of one GD1 molecule, 15 DMPC (7 in the upper layer which con-tains GM1 and 8 in the lower layer) and 539 water molecules. The other system (con-trol) had 16 DMPC molecule (8 in the upper layer and 8 in the lower layer) and 539water molecules. The energy of both the systems were minimized using conjugate gra-dient method in two stages. In the first stage all water molecules were kept fixed andonly the bilayer molecules were allowed to move. In the next stage only the water mol-ecules were allowed to move while the bilayer molecules were held fixed. The energyminimization were done for 20000-40000 steps with no non-bonded cutoff until therms deviation of energy was less than 0.001 kcal/mol.

The minimized structures of the two systems were then subjected to dynamics sim-ulation at constant volume and constant temperature of 300°K for 120 ps initialequilibration constraining all bonds using Rattle. Following the equilibration phasea dynamics simulation run of 400 ps was carried out. The time step was 2 fs. Thetime-averaged properties were calculated from the 400 ps trajectory using theDECIPHER module of InsightII.

Results and Discussion

GD1a Conformation and Environment

After elaborate equilibration of 120 ps the trajectory was recorded for 400 ps. Theresults presented here are from the 400 ps trajectory. A number of hydrogen bonds

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MD Simulation of GD1a-DMPC Bilayer

Figure 1: Structure of GD1a hexasaccharide.

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that are formed intra-molecularly of GD1a are listed in Table I. These hydrogenbonds were retained for the most of the times during the simulation. However therehad been a number of transient hydrogen bonds which are formed and lost. Severalhydrogen bonds are formed with the neighbouring solvent molecules also (data notshown). The carboxylate group of the external sialic acid is hydrogen bonded withthe hydroxyl OH4 of the external galactose. The amide (NH2) group of N-acetylgalactosamine is hydrogen bonded with the carboxylate group of the sialic acidwhich is attached to the internal galactose. NMR studies by Sabesan (1991)revealed the existence of such bond. The OH6 and O5 of the internal galactose arehydrogen bonded to the O5 atom of N-acetyl galactosamine and the hydoxyl OH3of the glucose respectively. The time series of the dihedral angles of the glycosidiclinkages of GD1a were calculated. Our calculated conformation of GD1a has astrong resemblance with the conformer found by Acquotti et al. (1994) (15 ) whichthey named as conformer 3. It is evident from Table II that most of the dihedral

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Figure 2: Starting configuration (the simulation box)for the GD1a+ bilayer simulations.

Donor Acceptor Distance AngleGD1a:G104H:H6 GD1a:G104H:O4 1.79 155.27GD1a:G104H:HO4 GD1a:H108H:O1A 1.69 175.83GD1a:G105H:HN2 GD1a:G108H:O1B 2.37 165.13GD1a:G106H:H6 GD1a:G105H:O5 2.17 166.93GD1a:G107H:HO3 GD1a:G106H:O5 1.96 161.79GD1a:G108H:HO4 GD1a:G108H:O10 1.72 172.14

Table IHydrogen bonds involving GD1a.

The residues are named as external galactose: G104H; external sialic acid: H108H; N acetylgalactosamine: G105H; internal galactose: G106H; glucose G107H; internal sialic acid:G108H respectively.

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angles are in a good agreement with the conformer 3. Changes are seen in the φ, ϕangles of the external sialic acid which was also found to fluctuate considerably inthe experiment. In the time averaged conformation of GD1a hexasaccharide bothsialic acid residues are oriented almost perpendicular to the plane of the neutralsugars. The internal branched sialic acid comes closer to GalNAc residue whereasthe external sialic acid extend more into the solvent.

The two hydrocarbon chains, the stearic acid and sphingosine chains of GD1aremain inserted into the hydrophobic core of the membrane through out the simu-lation period. In order to study the movement of the lipid tail of GD1a the meansquare displacements (MSD) of terminal carbon atoms of stearic acid and sphin-gosine both in the membrane bound form and free form were calculated. Data fromfirst 372 ps of 400 ps trajectory have been used for all MDS calculations. Meansquare displacements value of the individual atoms / groups were obtained as

MSD (t) = < |r (t) - r (0)|2 > [1]

The angular bracket in equation 1 imply averaging over the entire trajectory. Figure3 shows the MSD plots. It is evident from the plots that in the presence of the mem-brane the stearic acid movement is more restricted than sphingosine.

The aqueous environment of the GD1a atoms was examined by calculating radialdistribution functions [g(r)] which were obtained as

g(r) = N(r)/4πr2 ρ0 [2]

where N(r) is the number of water molecules between r and r + δr from the refer-ence atom. ρ0 is the bulk water number density (taken as the number of water mol-ecules per volume of the computational box). The water pair distribution functionof C of carboxylate group of external sialic acid and internal branched sialic acidsare given in Figures 4A and 4B. It is evident from the plots that the terminal sialicacid is more solvated than the internal branched sialic acid. There are about 10 mol-ecules of water within 8Å of external sialic acid and about 7 molecules of waterwithin 8Å of internal sialic acid respectively. The g (r) plots of O and N atoms of

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MD Simulation of GD1a-DMPC Bilayer

Sugar linkage Angle Trajectory Avg.(Using IUPAC nomenclature)*

Trajectory Avg.(Using NMR Convention)*

NMRconformation

Gal(β1-3)GalNAc φ -133.00 -14.84 -27.00(External) ψ -140.00 -22.53 -22.00

GalNAc((β1-3)Gal φ -91.82 27.24 30.00(Internal) ψ -104.41 15.59 20.00

Gal(β1-4)Glc φ -64.30 55.23 52.00ψ 116.68 -2.17 -1.00

Sia(α2-3)Gal φ 36.49 -90.29 -71.00(External) ψ -162.14 -46.34 1.00

Sia(α2-3)Gal φ -24.75 -147.94 -161.00(Internal) ψ -136.53 -18.45 -29.00

Table IIComparison of Sugar Conformation in GD1a.

or IUPAC nomenclature: φ = O5-C1-Ox-Cx, ψ = C1-Ox-Cx-C(x-1)

sialic acids:φ = O6-C1-Ox-C3, ψ = C1-Ox-Cx- C(x-1)

Where x is 3, 4 for the (1-3) or (1-4) linkage respectively.For NMR convention:φ = H1-C1-Ox-Cx, ψ = C1-Ox-Cx-Hx

sialic acids:φ = C1-C2-Ox-C3, ψ = C2-Ox-C3-H3Where x is 3, 4 for the (1-3) or (1-4) linkage respectively.*Values of the dihedral angles of GD1a obtained using NMR convention are also included for an easy comparison ofthe computed trajectory average conformation with the NMR conformation revealed by Acquotti et al. (1994).

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the acetyl group of terminal and internal sialic acids are given as Figures 4C, 4D,4E, 4F which also indicate that the acetyl group of the external sialic acid is moresolvated than the internal branched one. The above results indicate that there canbe differences in dynamics, solvation, conformation within GD1a.

The Polar Head Groups and Hydrocarbon Chains of the Bilayer

The lipid head groups mostly remain extended into the solvent. In order to studythe dynamics of the polar head group the mean square displacements (MSD) ofnitrogen atoms of the N(CH3)3 residue were calculated. The mean square plots are

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Figure 3: Mean square displacement (MSD) plots ofthe C atoms of: (A) Stearic acid in free form (B) Stearic acid in mem-brane bound form. (C) Sphingosine in free form (D) Sphingosine inmembrane bound form.

Figure 4: Radial distribution functions of water withrespect to different atoms in GD1a.(A) C of COO- of external Sialic acid.(B) C of COO- of internal Sialic acid.(C) N of acetyl group ( CH3CONH) of external Sialicacid.(D) N of acetyl group (CH3CONH) of internal Sialicacid.(E) O of acetyl group ( CH3CONH) of external Sialicacid.(F) O of acetyl group (CH3CONH) of internal Sialicacid.

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shown as Figures 5A and 5B. These figures show that the motions of the headgroups in the upper layer are similar to the motions of the head groups in the lowerlayer except for the lipid head group of the upper layer which is located next toGD1a. This head group moves more than rest of the N atoms of the polar headgroups. GD1a hexasaccharide being a bulky moiety induces a local disorder in thespatial arrangement of the polar head groups.

The dynamics of the hydrocarbon chains are also affected by the presence of GD1a.Although the overall pattern of the bilayer is retained, the GD1a induces localchanges. Figures 6A and 6B show mean square displacement (MSD) values of theterminal carbon atoms of the acyl chains. It can be seen from the figures that themovement of the lipid chains are almost similar for both upper and lower layers.Here also the neighbouring lipid chains from both the layers move significantlymore compared to the non-neighbour ones. This kind of local alteration in lipidarrangement has been observed by Berneche et al (1998) (35) where they foundthat the presence of melittin in the upper layer of DMPC bilayer has created avacant space in the middle of the bilayer. The acyl hydrocarbon chains from boththe layers adjacent to GD1a have much freedom in their movement as those havesome empty space to travel. The chains in the lower layer also adopted a moreextended configurations in an attempt to fill the gap. No such disorder wasobserved in the "bilayer-only" simulation (data not shown).

Conclusion

Our current study provides an effective method for exploring atomic details ofmembrane-GD1a interactions. The time average conformation of GD1a revealsthat the terminal sialic acid is more exposed into the solvent than the internalbranched one. Many interresidual contacts between N-acetyl galactosamine-inter-nal branched sialic acid; external gal-external sialic acid; N-acetyl galactosamine-internal gal are also observed. The conformation of the GD1a-hexasaccharide isstabilized by a number of intra molecular hydrogen bonds that were previously

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MD Simulation of GD1a-DMPC Bilayer

Figure 5: Mean square displacement (MSD) plots ofthe N atoms of (CH3)3N of polar head group. (A)Upper layer (B) Lower layer.

Figure 6: Mean square displacement (MSD) plots ofthe terminal carbon of the hydrocarbon tail. (A)Upper layer (B) Lower layer.

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observed experimentally. Though the overall arrangement of the bilayer is main-tained during the simulation, the presence of a single GD1a molecule has localeffects on the bilayer. A local disorder in the arrangement of the acyl chains as wellas the head groups is evident in the upper layer due to the presence of GD1a.

Acknowledgement

The work is funded by the Council for Scientific and Industrial Research,Government of India (No. 01(1500)/98/EMR-II). The use of the MolecularModeling Facility of the Bioinformatics Centre, Bose Institute, Calcutta andComputational Facility of the Computer Centre, University of Calcutta is grateful-ly acknowledged.

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M.H., Davis, S.J., Barclay, A.N & Dewk, R.A. (1999), Glycobiology, 9, 443-458.32. Siebert, H.C., Reuter, G., Schauer, R., von der Lieth C.W. & Debrowski, J. (1992),

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Date Received: October 9, 2000

Communicated by the Editor Rick L. Ornstein

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