Indian Journal of Biochemistry & Biophysics Vol. 38, February & April 200 I, pp. 96-103

Molecular modelling of MHC class I carbohydrates

Tarun K MandaI and Chaitali Mukhopadhya/ Department of Chemistry, University of Calcutta, 92, APC. Road, Calcutta 700009, India

Accepted 3 October 2000

In thi s article we present the results of molecular modelling of four complex carbohydrates which have been found in the MHC class I proteins. Though these proteins show di versity in their sequences, the glycosylation sites are hi ghl y conserved indi cating a possible structural/functional role of the glycan chain . Similar glycan chains have been found linked with other proteins of completely different fu nction, such as IgG, and erythropoeitin . Thus, the molecul ar modell ing of these carbohydrates will not on ly prov ide structural/dynamic information of these complex molecul es but will also provide conformational information which can be utili sed to build the glycoprotei n model s. The resul ts presented here indicate that although severa l linkages show less conformational nexibil ity, terminal linkages can be quite tlex ibl e.

Carbohydrates are the most abundant class of biomolecules. Bes ides being the source of energy, they have a variety of biological roles in storage and recognition l

-3. Complex carbohydrates occur 111

conjugated forms, such as glycoproteins or glycolipids, where the glycans are covalently linked to proteins or lipid molecules respectively. The glycans have an enormous structural diversity arising out of the differences in number, sequence, branching, glycosidic linkages and anomeric fo rm of the monosaccharides. Along with their structural heterogeneity the glycoconjugates are often found in different glycoforms, which means that the same protein or lipid can be glycosylated with different complex carbohydrates. Thus the contributions of these glycan chains on the structure and/or function of the glycoconjugates are difficult to establish3

-5

• The understanding at the molecular level of how the covalently linked sugar chains contribute to the properties of the glycoconjugates, requires the knowledge of the conformation of the linked carbohydrate chains.

The major histocompatibility complex (MHC) class I heavy chain protein is a glycoprotein containing a conserved glycosylation site6

•7. The

MHC class I heavy chain is associated with four types of related glycoforms7 (Fig. 1). Among the four glycoforms, one is triantennary and others are

* Author for correspondence Email: chaitali @cucc.ernet.in Abbreviations used: MHC, major hi stocompatibi lity complex; MD, molecular dynamics; nns, root-mean-square.

biantennary. Two of these glycan chains have terminal sialic acid residues. These type of complex N-linked glycans have been found to be attached with other proteins as well , like IgG, eryrhropoeiti n and tissue plasminogen activator4

.8

. It ha been reported that the glycan chain initiates the binding with the calnexin in endoplasmic reticulum and facilitates the assembly of MHC class I molecules and their the delivery to the cell surface9

.1O

. The recognition of the MHC class I molecules by antibodies and T cells is indifferent to the carbohydrate chains6

• Therefore, to understand the structure/function of MHC class I molecules at the atomic level, the kn wledge of the structure of glycan chain is important. The X-ray crysta. structure of MHC class I glycoprotein, however, does not contain any coordinates fo r the glycan chains6

.7.

There had been reports on the structure and dynamics of various oligosaccharides containing similar linkages, using NMR, fluorescence and molecular dynamics techniques t

, II -18 . However, the addition or deletion of different linkages to the core pentasaccharide (Man3GIcNAc2) might lead to different overall conformation of the oligosaccharides. Hence, in order to gain information about the structure and dynamics of these carbohydrates as well as to generate suitable glycan models for the generation of different glycoforms of MHC, molecular modelling of these glycans were carried out. In this article, we present the molecular mechanics and dynamics calculations of these carbohydrates. The individual disaccharides have been modeled first to identify the lowest energy

MANDAL & MUKHOPADHYA Y: MOLECULAR MODELLING OF MHC CLASS I CARBOHYDRATES 97

minima and these minima were subsequently used to generate the oligosaccharide models. The molecular dynamics simulation of these carbohydrates provide detailed information regarding the conformational flexibilities of d ifferent linkages. We compared the time-averaged conformations of these carbohydrates and have found that the conformation of the core pentasaccharide is very similar in all the four carbohydrates, where as the antennae can have a larger conformational flexibility. The fact that these molecules can adopt multiple conformations might be

biologically significant.

Methods

Disaccharide grid search The disaccharides were built from monosaccharide

templates provided in InsightlI Biopolymer module according to the glycosidic linkages. The glycosidic

dihedral angles (<p and \If) were changed from 0° to 360° by rotating the angles at 30° interval. Each conformation thus generated, was mjnimized keeping

Oligosaccharide -1

Gall3( 1-4)GleNAcl3(l-2) (a) I

SAa(2-6)Gall3(I-4)GleNAcl3( 1-4)Mana(I-3) Fuca( 1-6) ~) I I

Manf( 1-4)GleNAcl3( 1-4)GleNAcl3l-R

SAa(2-6)Gall3( 1-4)GlcNAcl3( 1-2)Mana( 1-6) (c)

Oligosac.:haride -2

SAa(2-6)Gall3( 1-4)G leN Acl3( 1-2)Mana( 1-3) (a) I

Fuca(I-6) I

GleNACI3(I-4)Manf(I-4)GICNACI3(I-4)GleNACI31-R

SAa(2-6)Gall3( 1-4)GleNAcl3( 1-2)Mana( 1-6) (b)

Oligosaccharide -3

GlcNAcl3(I-2)Mana(I-3) Fuca(I-6) (a) I I

Manf( 1-4)G leN Acl3( 1-4)G leN Ac131-R

GleNAcl3(1-2)Mana( 1-6) (b)

Oligosaccharide -4

GleNAcl3(1-2)Mana(1-3) (a) I

ManI3(1-4 )GleN Acl3( 1-4 )GlcNAcl3l-R I

GlcNAcl3(1-2)Mana(1-6) ffi

R - ASN-86, SA = Sialic Acid, GlcNAc = N-Acetyl Glucosamine, Fuc = Fucose, Man = Mannose, Gal = Galactose. Definitions of the glycosidic dihedral angles ~,\j1 and 0):

~ = Os-C1-0.-Cx , \j1 = C1-O.-Cx-C('.I), 0) = 0.-C6-CS -C4

Where x is 2,3,4 or 6 for the (1-2) , (1-3), (1-4) or (1-6) linkages respectively and $ = 0 6 • -C2-06-~ , \j1 = C2-06-~-CS , 0) = 06-~-CS -C4 for the (2-6) linkage.

Fig. I-Oligosaccharides prescnt in the MHC class-I glycoprotcins [The dihedral angles have been defined]

98 INDIAN J BIOCHEM BIOPHYS, VOL 38, FEBRUARY & APRIL 2001

the dihedral angles fixed. All the energy minimization and molecular dynamics simulations presented here have been carried out using the DISCOVER module and CVFF forcefield of the InsightII package. This force field has been updated to contain parameters fo r carbohydrates J9

. From the grid search , the lowest energy conformations were identified and minimized without further constraints. From these minima, the lowest energy conformation was taken as the minimized structure of that disaccharide.

The energy is minimized using conjugate gradient method for 500-1000 steps with no non-bonded cutoff. A constant dielectric of value l.0 was used. The low energy conformations of the disaccharides were subjected to dynamics in vacuum at room temperature 300K for 20 at ps an initial equilibration and then followed by 150 ps dynamics run using constant dielectric of value 1.0 and no non-bonded cutoff. From the trajectory, the average dihedral angles of the glycosidic linkages were analyzed.

Oligosaccharide model building The oligosaccharides 1 to 4 were modeled by

joining the disaccaharides according to their glycosidic linkages. The energy of the oligosaccharides were minimized using conjugate gradient method of 5000-10000 steps with no non­bonded cutoff until the rms deviation of energy was less than 0.001 kcallmol.

The minimized structures of the oligosaccharides were subjected to dynamics in vacuum at room temperature 300K for 20 ps initial equilibration, followed by 500 ps dynamics run using constant dielectric of value 1.0 with no non-bonded cutoff. From the trajectory (0.5 ps interval), the dihedral angles of the glycosidic linkages and their fluctuations were recorded. The conformational flexibili ties of the four oligosaccharides were compared.

Results and Discussion The molecular mechanics and dynamics

calculations were carried out using the DISCOVER module and CVFF forcefield of the Insight II package. This force field has been updated to contain parameters for carbohydrates J9

• It has been shown to generate realistic models of carbohydrates J2

, 13

Though results of similar carbohydrates containing some of the linkages, have been reported before J2

.J5

,

the effect of the exact composition and linkage patterns on the carbohydrates may be significant.

Hence, al l the four oligosacchardies have been modeled starting from the disaccharide templates.

The glycosidic dihedral angles (CI), '¥) (Fig. 1) of different di saccharides were varied from 0° to 3600 in 30° interva l generating 144 conformations. For the 1-6/2-6 linkages the <P, '¥ and ()J angles were similarly varied resulting in 1728 conformations. The energy minimization of all these conformations were done with fixed glycosidic dihedral angles. These minima were further relaxed by minimization with no constraints and the resulting conformations were used as the starting conformations for the molecu lar dynamics simulations of disaccharides as well as for the generation of the ol igosaccharides. The conformational analysis of individual di saccharides are listed below.

Mana( 1-3 )Man{3 From the grid search, the lowest energy

conformation was identified at 150°, -90°. The structure is further minimized without constrain and it relaxes to 150°, -99°. The energy difference between the fixed and relaxed conformation is 0.1 kcallmole. The lowest energy minima is subjected to vacuum simulation for 150 ps. The time series for the glycosidic angles were calculated from the simulated conformations. The average angles were 84°, -1 23°. The rms fluctuations were 29° and 36° respectively. It was also shown from the trajectory that the angles traversed through different local minima and this suggest that the linkage is flexible. This is in agreement with the previous observations by other authors I2

.15

• The minimum energy conformations of this disaccharide were used to construct the oligosaccharides 1 to 4. These oligosaccharides were minimized to remove any steric strain. The MD average conformation of the disaccharide linkage in the oligosaccharides-l, 2, 3 and 4 were 164°, -94°; 71°, -128°; 105°,77° and 80°, -109" respectively. The corresponding rms. deviation were 8°, 10°; 86

, 10°; 19°, 13°and 22°, 30° respectively . The average conformation of this linkage is qui te different in glycans 1 and 3 , whereas in 2 and 4 it is similar. The flexibility also decreases much in oligosaccharide-I, 0ligosaccharide-2 and 0Iigosaccharide-3. But in 0Iigosaccharide-4, the fl exibili lY was about the same as that in free disaccharide.

GlcNAc{X 1-4 )GlcNAc{3 The minima was obtained at _90°, 61°. After

relaxation, the energy conformation obtained was

MANDAL & MUKHOPADHYA Y: MOLECULAR MODELLING OF MHC CLASS I CARBOHYDRATES 99

-85°, 91°. The MD average conformation was -89°,

90° and during dynamics simulation, the rms

fluctuations in the dihedral angles were 29°, 17°. The

conformation of the disaccharide in the minimized

conformation of all these four oligosaccharides were

changed as shown in Table I. It was seen that the

isolated disaccharide conformation was drastically

changed in 0ligosaccharide-1 i.e. to 24°, 87°. Similar

conformation for this linkage has been reported

previollsl/ 2• The rms fluctuation of this glycosidic

linkage is 28°, 17°. The time series of this dihedral

angle (Fig. 2) indicate possible transition to other

minima. The disaccharide minima was retained in all

the other oligosaccharides. The MD average structure

of this linkage in oligosaccharides-2, 3 and 4 were -

81°, 107°; -66°, 122° and -90°, 88° respectively. The

corresponding rms fluctuations were 16°, 15°; 9°, 11°

and 33°, 15°.

GlcNAc{3{ 1-2)Mana The lowest energy conformation obtained from the

grid search for GIcNAc~(l-2)Mana. was -85°, -155° which on further minimization relaxed to -86°, -135°. The energy difference between these two conformations was about 0.6 kcallmole. The MD average conformation of this disaccharide was -81°, -122°. The rms. deviation was 15°, 19°. This disaccharide occurs at two antenna of the oligosaccharide. The trajectory average conformations are given in Table 1. As can be seen from the Table, that apart from the smallest glycan 4 and in the (1-3) arm of glycan 3, in all the other glycans the flexibility of this linkage is less and the conformation is also similar. Therefore, the flexibility decreases In

oligosaccharide-l and 2 and (1-6) antenna of oligosaccharide-3. In contrast, it increases In

oligosaccharide-4 and (1-3) antenna of oligosaccharide-3. This result suggests that multiple

Table I-Conformation of the glycosidic linkages in the disaccharides and oligosaccharides [*The numbers within the brackets () indicate the rms Ouctuations during dynamics simulations]

Mana( 1-3)Man~ Oligosaccharide-I Minima MD Minima MD

150 -99

average

84(29) -123(36)

GIcNAc~( 1-4)GlcNAc~

170 -82

-85 -89(29) 24 91 90(17) 87

GIcNAc~( 1-2)Mana -86 -81(15) (a)

-135 -122(19) -97

Man~( 1-4 )GIcNAc~

-141 (c) -73 -83

-64 -89(39) -81 109 90(13) 101

Gal~( 1-4)GIcNAc~ -76 -72(14) (a) 107 109(13) -84

98 (b) -124 98 (c) -91 100

average

164(8) -94( 10)

12(28) 92( 17)

(a) -96( II) -135(7)

(c) -72(11) -91(13)

-78(8) 106(8)

(a) -87(11 ) 99( 10) (b) -82(9) 105(9) (c) -116(14) 86(9)

01 i gosacc haride-2 Minima MD

average

70 71(8) -135 -128(10)

-98 -81 (16) 87 107(15)

(a) (a) -73 -74(11)

-128 -133(12) (b) (b)

-101 -98(9) -82 -89(11 )

-79 -77(8) 114 110(10)

a) a) -71 -70(12) 97 103(10) (b) (b) -98 -101(9) 80 76(9)

Oligosaccharide-3 Oligosaccharide-4 Minima MD Minima MD

average average

62 105(19) 62 78(22) -141 77(13) -140 -109(30)

-94 -66(9) -78 -90(33) 92 122(11) 99 88( 15)

(a) (a) (a) (a) -97 -78(27) -97 -74(29) -98 -106(26) -97 -113(36) (b) (b) (b) (b) -85 -89( II) -93 -62(32)

-134 -117(17) -143 -62(27)

-66 -64( 15) -58 -36(28) 119 108(9) 122 105(12)

100 INDIAN J BIOCHEM BIOPHYS. VOL 38. FEBRUARY & APR IL 2001

(2) Psi

~~

0 ......... cO. 0

.--i

I '

(lJ --' (J) 0 (3) c co « .-I

o 500 o 500

Time(ps) Fig, 2-Time series of the GlcNAcf)(1-4)GlcNAcf) dihedral angles <!>(phi). \jI(psi) fluctuations of oligosaccharides I to 4 [0) to (4) respecti vel y I

conformations are possible for this linkage and the flexibility depends on the location and nature of the antenna.

Manf3{ 1-4 ) GLcNAc{3 The lowest energy conformation _49°, 98° was

identified from the grid scan search and further minimized without constraint. The relaxed conformation was obtained at 64°, 109°. The energy difference between this two conformation was - 0.2 kcallmole. The MD average conformation of this disaccharide was -89°, 90° and rms fluctuation was 39°, 13°. In oligosaccharides-l, 2, 3 and 4 the respective MD average conformations were -78°, 106°; _77°, 110°; _64°, 108° and -36°, 105° with corresponding rms deviation of 8°, 8°; 8°, 10°; 15°, 9° and 28°, 12°. This indicates that the flexibility decreases in oligosaccharides-l, 2 and 3 whereas, it increases to some extent in 0ligosaccharide-4 which is the smallest in size.

GaLf3{ 1-4 ) GLcNAc{3 The grid search was carried out to identify the

lowest energy conformation and obtained at -90°,91°. This structure was further minimized removing the constraint. The glycosidic linkage relaxed to -76°, 107°. The energy difference betw'een these two conformations was ].2 kcallmole. The MD average angle of this glycosidic linkage was ··72°, 109°. The rms deviation of this linkage was 14°, 13°.This linkage is present in glycans-l and 2 only. The generated oligosaccharides were minimized and results are shown in Table ]. The average conformation and flexibility of this Unkage was more or less same in both the glycan .

GLcNAcf3{ 1-4 )Man{3 This linkage is present in the oligosaccharide-2

only. The grid scan search provided the lowest energy minima at -70°, 100°, which upon further

MANDAL & MUKHOPADHYA Y: MOLECULAR MODELLING OF MHC CLASS I CARBOHYDRATES 101

Table 2-Conformation of the a(2-6) and a( 1-6) glycosidic linkages in the disaccharides and oligosaccharides [*The numbers within the brackets ( ) indicate the rms fluctuations during dynami cs simu lations]

SAa(2-6) Galp Minima

86 -78

-169

MD average

170(30) -162(34) 174(12)

01 igosaccharide-I

Min ima MD average

(b) (b) -177 141 (50) 142 163(31 ) 177 -164(34)

(c) (c) - 170 - 172(9) - 137 - 140(9) - 167 - 172(9)

0ligosaccharide-2 Minima MD average

a) a) 168 127(50)

-161 -176(26) 177 -1 74(15) (b) 178 (b)

-133 -178(8) 174 - 136(12)

173( 10)

Fuca( 1-6) GlcNAcp Oligosaccharide-I 01 i gosaccharide-2 0ligosaccharide-3

Minima MD average

-75 -89(32) -91 -170(32) -58 -60( I 0)

Mana( 1 -6)Man~

Minima

-62 178 -60

Oligosaccharidc-I Minima MD Minima MD

average average

66 75 (14) 144 11 6( 12) -168 - 136(18) 74 79(8) -175 -169( I 0) 179 -179(6)

MD Minima average

-79(23) -143 -153(43) -165 -88(73) -72

0ligosaccharide-2 Minima MD

average

69 75(11) 68 70(8) 175 175(6)

(c)

MD Minima average

-97(33) -72 -177(41) -86 -160(35) -72

0ligosaccharide-3 Minima MD

average

68 78( 18) -172 73( I 0) - 166 166(8)

MD average

-96(34) 162(3 1) 50(10)

0ligosaccharide-4

Minima MD average

64 91(24) -169 -88(54) 169 - 164(14)

Fig. 3-Supcrimposed snapshots from trajectory [The snapshots were taken at every 100 ps interval (a) oligosaccharide- I, (b) oligosaccharide-2, (c) oligosaccharide-3 and (d) o li gosaccharide-4J

102 INDIAN J BIOCHEM BIOPHYS, VOL 38, FEBRUARY & APRIL 200 1

mInllTIl zation went to -730, 1 100

, In the 0ligosaccharide-2 it adopted an average conformation of -59°, 125° during the dynamics simulations wi th rms fluctuati ons of 100

, 80,

The a(2-6) and a( 1 -6) linkages The resul ts of the di saccharide grid search and

minimization/dynamics study of the di saccharides and the oligosaccharides are listed in Tab le 2. It is interesting to note that the SAa(2-6) linkages in oligosaccharides-l and 2 in the a(l-6) arm is more flexible compared to th at in the a(l-3) ann . The Fuca(l-6) linkage in 1, 2 and 3 show sign ificant flexibility with higher rms fluctuations compared to the other lin kages. The Mana(l-6) linkage, on the other, hand shows cons iderable rigidity In oligosaccharides-l, 2 and 3.

O/igosaccharides -1,2, 3 and 4 The o li go.~ accharides were generated by joini ng the

01 l'-l'--

---- C') <t

-I-'

lfl

0

C\l

lD

'<t ,--;

,--;

lD l'--

lowest energy minima of the necessary disaccharides. The generated oligosaccharides were energy minimi zed to remove any steric strain . The conformation of the linkage dihed ral angles were noted (Table I and 2). Some of the minimum energy conformati ons of the disaccharides got modified in the oligosaccharides. The mini mized conformations of the oligosaccharides were subjected to 500 ps dynamic run . The time series of the dihedral angles of glycosidic linkages were calculated from the dynamics trajectories. The average conformation and the rms fluctuations of the dihedral angles are li sted in Tables I and 2.

The average conformation of these oligosaccharides contain a large number of inter­residue hydrogen bonds which stabili ze the oli gosaccharide conformations (data not shown). It is evident from Tables 1 and 2 that the in ternal glycosidic linkages were relatively rigid compared to the terminal ones. Apart from a few diffe rences, the

C')

OJ

((l (4 C\l

OJ

Tim e(ps)

Fig. 4-Time series of the d is tances between Man~C3 and sialic acid C7 in ( 1-3) arm and between Man~C5 and sial ic acid C7 in (1-6) arm of the oligo- l 1( 1), (2)J and oligo-2 1(3), (4)1

MANDAL & MUKHOPADHYAY: MOLECULAR MODELLING OF MHC CLASS I CARBOHYDRATES 103

core pentasaccharide Man3GlcNAc2. retains similar conformation. In glycan 1, core GlcNAc~(1-

4)GlcNAc~ linkage undergoes some conformational transitions (Fig. 2) which is also evident from its increased fluctu ations (Table 1). The average conformation of thi s linkage is also quite different from that in the other glycan chains. It was found that this residue is hydrogen bonded to the Mana residue on the (1-6) arm. This hydrogen bond stabilizes the altered conformation of this oligosaccharide. Similar conformation of this linkage has been reported earlier for a related glycan chain 12.

It was found that if superimposed on to each other using the core pentasacchride then the rms deviation between the average oligosaccharide conformations range from 1.8 to 2.5 A which indicates an overall similarity of the core region. The 0ligosaccharide-4 containing the least number of branching shows significant structural flexibility which is reduced in the other three oligosaccharides. Superimposed snapshots of the oligosaccharides from the individual trajectories are provided in Fig. 3a to 3d. It is seen from the figures that though the core is rigid, there is a considerable flexibility in the conformation of the antennae. In both 0ligosaccharides-3 and 4 the (1-3) and (1-6) arms containing only GlcNAc and Man residues (Fig. 1) are quite flexible. Whereas increasing the arm length by introducing Gal and sialic acid residues restricts the movements of the arms to some extent as in oligosaccharides-l and 2. In these oligosaccharides sialic acid residue on the (1-3) arm shows increased flexibility compared to the same linkage in (1-6) arm. The average end-to-end distance of the two arms in the two glycans was calculated as the distances between the central Man~C3 to sialic Acid C7 for the (1-3) arm and Man~CS to sialic acid C7 for the (1-6) arm. The Fig. 4 shows the variation of these distances in the two glycans highlighting that in oligosaccharide-l the (1-6) arm is more stretched than the (1-3) arm whereas, in 0ligosaccharide-2 the (1-3) arm is more stretched.

The presence of a bifurcating GlcNAc residue at the fourth posItion of the central Man~ in 0ligosaccharide-2 changes the conformation of the Mana(l-3) and Mana(l-6) linkages to the central Man~ (Table 1) as compared to those in oligosaccharide-I. The 0ligosacchraides-3 and 4 differ in only the terminal Fuca(1-6) attachment. It has been seen that the lack of Fuca(1-6) linkage in

0ligosaccharide-4 induces conformational flexibility

to the terminal GlcNAc~(l-4)GlcNAc~ linkage, which has a rms fluctuation of 9°, 11° and 33°, 15° respectively in the 0ligosaccharides-3 and 4 respectively.

The biological significance of the conformations of complex carbohydrates might arise from their function as carriers of important information. The results presented here indicate that various conformational possibilities are available for these oligosaccharides. The flexibility of the same glycan linkage is affected depending upon the extent of chain length and branching. That the same glycan chain could be attached to proteins with diverse functions suggests that the same glycan chain can be utili zed differently by different proteins and the conformation of the same glycan chain in different protein chains might be different.

Acknowledgement This work was supported by the Council of

Scientific and Industrial Research (CSIR), Government of India (No. 01(1S00)/98/EMR-II).

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