Molecular Dynamics Simulation of Lewis Blood Groups and Related Oligosaccharides CHAlTALl MUKHOPADHYAY and C. ALLEN BUSH Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21 228

SYNOPSIS

Molecular dynamics simulations without explicit inclusion of solvent molecules have been performed to study the motions of Lewis" and Lewisb blood group oligosaccharides, and two blood group A tetrasaccharides having type I and type I1 core chains. The blood group H trisaccharide has also been studied and compared with the blood group A type I1 core chain. The potential energy surface developed by Rasmussen and co-workers was used with the molecular mechanics code CHARMM. The lowest energy minima of the component disaccharide fragments were obtained from conformational energy mapping. The lowest energy minima of these disaccharide fragments were used to build the tri- and tetrasaccharides that were further minimized before the actual heating/equilibration and dynamics simulations. The trajectories of the disaccharide fragments, e.g., Fuc a-(1+ 4)GlcNAc, Gal 0-(1 + 4 ) GlcNAc, etc., show transitions among various minima. However, the oligosaccharides were found to be dynamically stable and no transitions to other minimum energy conformations were observed in the time series of the glycosidic dihedral angles even during trajectories as long as 300 ps. The stable conformations of the glycosidic linkages in the oligosaccharides are not necessarily the same as the minimum energy conformation of the corresponding isolated disaccharides. The average fluctuations of the glycosidic angles in the oligosaccharides were well within the range of f15O. The results of these trajectory calculations were consistent with the relatively rigid single-conformation models derived for these oligosaccharides from 'H-nmr data.

INTRODUCTION A class of small complex carbohydrates, known as blood group oligosaccharides, has been identified as the immunological determinants for the genetically inherited human A, B, and 0 blood types important in blood banking. Oligosaccharides isolated from epithelial mucins found in ovarian cyst fluids have been shown to inhibit reaction of antibodies both with red blood cells as well as with blood group active mucins secreted from the epithelial tissues in most subjects.' Defined tri- and tetrasaccharide fragments associated with blood group reactivity have been isolated from these ovarian cyst mucins, and their structures have been determined by classical chemical methods.2 Presumably these same oligosaccha-

Biopolyrners.Vol. 31, 1737-1746 (1991) CCC 0006-3525/91/141737-10$04.00 0 1991 John Wiley & Sons, Inc.

rides responsible for blood group specificity appear at the nonreducing terminals of either glycoproteins or glycolipids on the red blood cell surface, although direct chemical evidence on this point is incomplete. Although transfusion of blood having incompatible A, B, or 0 blood type causes strong hemolytic reactions, the underlying biological function of the blood groups is not known. A number of additional blood group determinants associated with different oligosaccharides are known that react only weakly and do not cause serious hemolytic response. The Lewis blood group determinants are in this class. Oligosaccharides with chemical structures closely related to blood group determinants, especially carbohydrates related to the Lewis type, have been implicated as tumor antigens and as differentiation markers3 Similar oligosaccharide structures related to the Lewis type have been shown to be receptors for leukocytes important in the inflamatory re~ p o n s eTherefore, .~ in spite of the uncertainty con1737

1738

MUKHOPADHYAY AND BUSH

cerning the general function of blood group carbohydrates, there is considerable interest in their conformation and dynamics inspired by the possibility of using these molecules or related analogues either as diagnostic tools or for therapeutic intervention in a number of significant biological processes. An important experimental technique for studying the conformation and dynamics of oligosaccharides is 'H-nmr spectroscopy, and in particular 'H nuclear Overhauser effect spectroscopy ( NOESY ) data have proved to be a most useful approach. Evidence based on this technique has been presented that oligosaccharides of the asparagine-N-linked giycoproteins exhibit multiple conformation^.^ Similarly, nmr studies of the oligosaccharide chains of gangliosides argue for exchange among several related conformations of these important membrane glycolipid constituents.6 In contrast to the evidence for considerable flexibility in some classes of oligosaccharides, studies by Lemieux and co-workers on blood group oligosaccharides led to the proposal that single conformations dominate in these carbohydrates.' Subsequent nmr experiments in our laboratory have supported the proposal of Lemieux and co-workersproviding additional evidence for the idea that the conformations are determined mainly by steric r e p u l ~ i o n . ~These ,~ results suggest that complex oligosaccharides of glycoproteins and glycolipids might be divided into groups characterized by their conformational flexibility with some oligosaccharides relatively rigid while others may have identifiable L'hinge"' or points of flexibility." In all these nmr studies of oligosaccharide conformation, molecular modeling is an important component without which interpretation of the data would be impossible. The earliest models were based on so-called rigid-geometry methods employing internal coordinates, in this case the glycosidic dihedral angles $ and $. The use of internal coordinates reduces the number of degrees of freedom to a minimum, making possible exhaustive searches of the oligosaccharide conformational space at least for oligosaccharides of modest size. More recently, methods of molecular modeling in which all the degrees of freedom are considered have been introduced. In this approach, an exhaustive search is impractical and a particularly efficient means for handling the large numbers of degrees of freedom is to solve Newton's equations in Cartesian coordinates for the motion of the atoms. This "molecular dynamics (MD ) method" has recently been applied to carbohydrates with considerable success.11 The method of MD simulations has been applied to blood group oligosaccharides, providing some further in-

sight into the interpretation of the nmr data on these relatively rigid structures.12 In the present article, we describe the extension of these MD studies to additional structures of the blood group class, including the Lewis blood group oligosaccharides.

METHODS All the calculations reported here were performed with CHARMM version 21 running on Silicon Graphics workstations. The Newton's equations of motions were integrated using Verlet a l g ~ r i t h m ' ~ s. The bond lengths with a step size of 5 X were kept fixed using the SHAKE14algorithm with an error tolerance of lop6. These studies employed the potential energy surface developed by Rasmussen and co-worker~.'~ The choice of this force field was made on the basis of the fact that this parameter set has some condensed phase information built into it in an average way. Hence for the present type of simulations in which a single molecule in vacuum is being studied, this parameter set is the more suitable choice over the CHARMM-like parameterization,16 where large values of the hydroxyl atomic partial charges are used to adequately treat the hydrogen bonding. The later set of parameters, then, are more appropriately used in simulations in which solvent or crystal environments are explicitly included. The parameters for the atoms of the amide group of the acetamido sugars were taken from the CHARMM parameter set. Although these two parameter sets are not strictly compatible, the unavailability of one consistent force field made this inclusion necessary. The molecules studied are the Lewis" and Lewisb blood group oligosaccharides and two blood group A tetrasaccharides having type I and type I1 core chains. The structures of these sugars are given in Table I. The QUANTA software package and its residue topology files were used to generate the molecules. Some of the topology files were modified to meet our purposes. All the monosaccharides units are D sugars and were generated in their 4C1conformations excepting Fucose, which is an L sugar, and it was generated in the 'C, conformation. The glycosidic dihedral angles 4 and $ are defined as Oring-Cl-Ol-Ciand Cl-Ol-Ci-Ci-l, respectively, following IUPAC conventions. The amide dihedral angle 7 is defined as Cl-C2-N2-C. The disaccharide units were studied first to choose their lowest energy minimum by generating their conformational energy maps. These maps were obtained by varying the two glycosidic dihedral angles independently from -180'

MD OF LEWIS BLOOD GROUPS

1739

Table I Structures of Blood Group Oligosaccharides Oligosaccharide Lewisd

Primary Structure Fuc a-(1+ 2)Gal p-(1+ 3)GlcNAc @-OMe

FUCa-(1+ 4)

Lewis"

Gal p-(1+ 3) 7 GlcNAc p-OMe

FUCa - ( l+ 4)

Lewisb

Fuc a-(1+ 2)Gal @-(I Type I A

7

GlcNAc /3-OMe

/*

Gal p-( 1 + 3)GlcNAc p-OMe

FUCa-(l + 2 ) \ GalNAc a-(1+ 3)

Blood group H

3)

Fuc a-(1 + 2) I GalNAc a-(1+ 3)

Type I1 A

+

7

Gal p-( 1 + 4)GlcNAc 0-OMe

Fuc a-(1-P 2)Gal P-(1 + 4)GlcNAc a-OMe

to 180" in 30" steps. The energy of the molecule at each step was minimized by relaxing all the degrees of freedom excepting the 6 and 1c/ angles. The lowest energy minimum of the disaccharides obtained from such maps was used to build the tri- and tetrasaccharide models. These resultant oligosaccharides were finally relaxed completely by energy minimization. These minimized structures were used as the starting structures for the subsequent MD simulations. The standard CHARMM minimizer ABNR was used for all the minimizations. The trajectories were then initiated by assigning the atomic velocities from a Maxwellian distribution at a low temperature (0-10 K ) and thermalized by increasing the temperature by 15" increments at 0.25-ps intervals until the desired value of 300 K was reached. The trajectories were equilibrated at this temperature by periodic scalings of the atomic velocities if the average molecular temperature drifted from this temperature by more than 5 K. For most of the trajectories the duration of the equilibration period was 20 ps but was extended by another 10 ps for a few difficult cases of equilibration. The simulation period started after the equilibration and the data collection was done without further intervention. During the simulation periods the energies of the trajectories were well conserved and no temperature scaling was necessary. There was no overall drift in the system temperature and the rms fluctuation was less than 15". The time series of various dihedral angles were calculated from the coordinate sets saved during the simulation period.

RESULTS Dynamics of Monosaccharides in Di-, Tri-, and Tetrasaccharides

In general the chair conformations ( 4C, for Gal and GlcNAc and 'C4 for Fuc) of the monosaccharide units were found to be dynamically stable. However, the conformations are not completely rigid; all the ring torsions undergo small fluctuations about the mean values during the simulations. This produces an overall oscillatory puckering of the ring with the average fluctuations generally in the range of 5"8". This range agrees with those obtained by Brady in the case of D-glucopyranoses using the same parameter set.17 However, it has been found that the GlcNAc residue in the Lewis tetrasaccharide undergoes transitions from chair to skew-boat or half-chair conformation during a 300-ps trajectory. No experimental evidence supporting such a transition has been obtained so far. The transition from the chair to the skew-boat conformation was modeled by changing the dihedral angle of C5-05-Cl-C2 at 5" intervals and relaxing the rest of the molecule. We found that for a rotation of 120" around the 05-C1 bond, the energy barrier is only 4 kcal/mol at 0 K. This low barrier of the Rasmussen potential function could be surmounted at higher temperature ( 300 K ) by the numerous thermal fluctuations that take place at this temperature. Similar spontaneous ring transitions have also been observed in the MD simula-

1740

MUKHOPADHYAY AND BUSH

tion of P-D-glucopyranose" and also in the vacuum dynamics of a-cycl~dextrin.'~ In the former case the same potential force field of Rasmussen and his coworkers had been used. In the later case, using the GROMOS force field, it was found that the fifth glucose residue, which is conformationally strained, underwent a flip. In the present case, the observed transition is thought to be unrealistic, since the homonuclear proton-proton coupling constant data do not indicate significant contributions from any forms of the pyranose ring other than the chair form. When this 300-ps trajectory for the Lewis * tetrasaccharide was repeated with the CHARMM-like force constants,I6 no transitions in the ring dihedral angle trajectories were observed. However, the nature of the time series of the glycosidic dihedral angles remained unchanged. This suggests that the force constants of Rasmussen potential functions are too low to arrest such transitions during relatively long simulations. No trajectories in which transitions from the normal chair conformations of any monosaccharide residue has been observed were used in the analysis of the oligosaccharide conformations. Dynamics of Disaccharides

Fuc a-(1 + 4)GlcNAcfl-Ome. Both glycosidic an-

gles were independently varied over the range of -180" to +180" a t 30" intervals. Th e resultant 144 structures were minimized by 100 steps of ABNR. The glycosidic angles were held rigid during the minimization and the rest of the molecule was

-180

0

0 0

180

0

9 Figure 1. Energy contour map of Fuc a-(1 --t 4 ) GlcNAc P-OMe, contours drawn at 1-kcal intervals.

150

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;

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Figure 2. Trajectory of disaccharide Fuc a-(1 4)GlcNAc P-OMe: (lower trace), (upper trace).

+

+

treated as flexible. I n the contour plot in Figure 1, the minimum energy conformation was obtained at rp = -90" and = 60". When this structure was further minimized, the glycosidic dihedral angles relax to 4 = -75" and 1c/ = 110", the conformation chosen as the starting conformation of this disaccharide moiety in the Lewis blood group oligosaccharides during the MD simulation. Four independent 40-ps simulations were started and one of them was extended to 120 ps. Time series for the glycosidic angles were calculated from the simulated conformations. A typical trajectory (Figure 2 ) showed evidence of conformational transitions as did all of the trajectories for this disaccharide. The rms fluctuations of the glycosidic angles during the 120-ps simulation were 16" and 25", respectively. Several times during the 120-ps simulation these angles have shown transitions to 4 = -70", $ = 140" and 4 = -140", $ = 100". When the first conformation was minimized, it converged to the same minimum with q5 = -75", $ = 110'. However, the second conformation minimized to a different minimum with 4 = -130", 1c/ = 88", showing that during the simulation the disaccharide actually traverses through different minima. This further suggests that considerable amount of flexibility exists in the glycosidic linkages of the disaccharide. Analysis of the motion of the side chain -NHCOCH3 showed that the torsion angle 7 fluctuates around two minima, 90" ( f 2 0 ) and 150" (+20) (Figure 3 a ) . The existence of these two minima are mainly governed by the nonbonded interactions between the pendant groups a t the C1, C2, and C3 positions.

+

Gal & ( 1 + 4)GlcNAc B-OMe. A grid scan search similar to th a t described above for Fuc a-(1 +

1741

MD OF LEWIS BLOOD GROUPS

blood group oligosaccharides, e.g., Fuc a-(1 + 2)GlcNAc, Gal p-( 1 + 3)GlcNAc, etc., has been reported previously." All of them show transitions among various minima.

160. 100.

50. I

v) 0,

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-22

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Figure 3. Trajectory of r in GlcNAc residue of ( a ) disaccharide Fuc a-( 1 + 4 ) GlcNAc, ( b ) Lewis' trisaccharide, and ( c ) Lewisb tetrasaccharide.

4 ) GlcNAc 0-Ome, has been carried out to identify the lowest energy minima of this disaccharide. The minimum was obtained a t 4 = -60", rC, = 120". This conformation was further allowed to relax to 4 = -56", # = 115". Three 40-ps simulations were started and one of them was extended to 120 ps (Figure 4 ) . The lack of conformational transitions in the trajectory indicates that this disaccharide is relatively more rigid than the Gal p- ( 1+ 3)GlcNAc studied by Yan and Bush." The dynamics of the other disaccharide fragments in the Lewis and A

Dynamics of Oligosaccharides Lewis" Trisaccharide (Gal 8-f 1 + 3 ) [ Fuc a- f 1 + 4 ) ] GlcNAc 8-OMe). The starting conformation of the disaccharide moiety Gal 0-( 1 + 3 ) GlcNAc p-

OMe was taken to be 4 = -60°, )I = -120", obtained from the grid scan search." The resultant Lewisa trisaccharide conformation was minimized and the minimum was obtained a t c$(Gal) = -70", rC,( Gal) - -105", and ~ ( F U C = )-72O, +(Fuc) = 137", the conformation chosen for starting the subsequent MD simulations. Three distinct 75-ps trajectories were started and one of them was extended to 300 ps. The trajectories (Figure 5 ) have shown only a few short-lived deviations from the minimum-energy conformation, suggesting that the glycosidic linkages of the Lewisa trisaccharide are much less flexible than the disaccharide fragments. The disaccharide Gal 0-( 1 + 3 ) GlcNAc P-OMe has been previously studied by Yan and Bush,12 and has been found to be quite flexible. The same linkage in the trisaccharide is quite rigid, the rms fluctuations of the 4,J. angles are 10",9", respectively. The Fuc a- ( 1 + 4) linkage in the trisaccharide is also considerably more

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Time in ps

T i m e in p s

Figure 4. Trajectory of disaccharide Gal p-(1 4)GlcNAc p-OMe: 4 (lower trace), $ (upper trace).

-+

Figure 6. Trajectory of Lewis" trisaccharide. ( a ) q5 Gal ~-(1+3),(b)J/Gal~-(l-+4),(c)q5Fuca-(1-+4), and ( d ) J/ Fuc a-(1+ 4 ) .

1742

MUKHOPADHYAY AND BUSH

rigid compared to the isolated disaccharide (cf. Figures 5 and 2 ) . The rms fluctuations of the 4 and $ angles are 13",9" in the trisaccharide and 16",25' in the disaccharide. It is interesting to observe that the average conformation of Fuc a- ( 1+ 4 ) GlcNAc P-OMe in the disaccharide (-93", 77") is different from that in the Lewis" trisaccharide (-71",142'). This conformation was attained by the a- (1 + 4 ) linkage during the process of minimization, and remained stable through out the equilibration and simulation period. This conformation of the a(1 + 4)linkage, which is prevalent in the trisaccharide, has also been observed in the disaccharide trajectory as a minor conformation and when minimized it actually merged to the global minimum. If the low-energy conformation of the disaccharide, Fuc a- ( 1+ 4) GlcNAc, is incorporated in the Lewis" trisaccharide, repulsive van der Waals interactions are observed between H5, C6, H4 of Fuc and 05, H2,04 of the Gal moiety attached at the C3 position of the GlcNAc ring. During the minimization of the trisaccharide, these overlaps are relaxed and the Fuc a- (1 + 4)linkage stabilizes at the $,$ values of -72', 137". The average values of the 4,$ angles of the different linkages and their rms fluctuations obtained from the 300-ps trajectory have been provided in Table I1 along with those obtained from the NOE experiments done on the nonreducing terminal Lewis blood group determinant fragments of the milk oligosaccharides, Lacto-N-fucopentaose-2 (LNF-2).' The comparison of the data shows a very

good agreement between the simulated and experimentally observed conformations. The conformational energy calculations on type 1 and type 2 pgalactosyl linkages 'O have yielded several low-energy conformations for the acetamido side chain at C2 of GlcNAc. Two of them were r = 100" and 140°, and the other was r = -60'. The one with r = -60" was ruled out by Cagas et aLZ1based on the coupling constant data. In our simulations, the minimum near 100' is more populated than the minimum near 140" (Figure 3b), showing that the addition of the Gal residue to the C3 position of the GlcNAc ring vicinal to the amide group restricts the movement of this side chain to some extent. Lewis Tetrasaccharide Fuc (Y- (7 + 2) Gal 8-(1 + 3)[ Fuc a-(7 + 4 ) ] GlcNAc 8-OMe. The minimumenergy conformation of the Fuc a- ( 1 + 2) linkage ( -60",150') obtained from the grid scan searchI2 was taken as the starting conformation of this moiety in the tetrasaccharide. The minimized conformation of the tetrasaccharide has the following @,$values:a- (1+ 2)linkage (-67",135"); P- (1+ 3)linkage (-67", -104"); and a - ( 1 + 4)linkage ( -82",140" ). This conformation was chosen as the starting conformation for simulations of the Lewisb tetrasaccharide. Three different 100-ps trajectories were started and two of them were extended to 300 ps. The time series of the glycosidic dihedral angles show that even on time scales as long as 300 ps the average conformation of the tetrasaccharide is dynamically stable and fluctuates around the minimum

Table I1 Average Conformations of the Oligosaccharides Obtained from the MD Simulations

Molecule Disaccharide Disaccharide Lewis" Lewisb

Type I A

Type I1 A

Blood group H

* Refs. 9 and 21.

Linkage Type

Average Value 4, J. (deg)

Fluctuation (ded

NOE Result* (ded

FUCa-(1+ 4) Gal p-(1 + 4) Gal p-(1 + 3) Fuc a-(1+ 4) Fuc a-(1+ 2) Gal p-(1 + 3) Fuc a-(1+ 4) Fuc a-(1+ 2) GalNAca-(1 + 3) Gal p-(1 + 3) Fuc a-(1+ 2) GalNAca-(1 + 3) Gal p-( 1 + 4) Fuc a-(1+ 2) Gal p-(1+ 4)

-93,77 -46,111 -72, -103 -71, 142.5 -75,138 -61, -111 -62,148 -66, 138 60, -164 -62, -112 -63,136 59, -163 -52,117 -71,141 -40,111

16.2, 24.9 20.0, 11.9 10.5, 7.7 13.5, 7.4 10.6, 9.7 8.9, 9.1 13.3, 9.7 7.7, 6.9 9.3, 11.7 8.6, 8.7 9.3, 7.5 12.2, 11.3 11.4, 8.9 11.7, 8.8 17.6, 12.1

-

-70(t10), -100(+10) -70(+10), 140(f10) -8O( tlo), 140(f10) -70(-t lo), - 100(f 10) -70(+10), 140(+10) -70(+10), 140(+10) 50(f10), -160(+10) -80(f10), -100(+10) -80 (+ lo), 140(f10) 60(+10), -160(&10) -9O(f10), llO(f10) -

MD OF LEWIS BLOOD GROUPS

-50. -100.

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250.

Time in ps

Figure 6. Trajectory of Lewisb tetrasaccharide. ( a ) (b F'UC a- ( 1 --* 2 ) , (b) fi FUCa-( 1-P 2 ) , ( c ) fi FUCa - ( 1 + 4 ) , ( d ) (b Fuc a-(1+ 4 ) , ( e ) Gal p-(1 + 3 ) , and ( f ) (b Gal p-(1+ 3 ) .

+

energy conformation (Figure 6). The average values and rms fluctuations of the 4 and $ angles are provided in Table 11. As in the case of the Lewisa trisaccharide, excellent agreement was found between the simulated and experimentally observed conformations of Lewis b . Analysis of the conformation of the amide side chain of the GlcNAc residue shows that, in the Lewis tetrasaccharide, the side chain fluctuates around a single minimum, 95" (f15") (Figure 3c). When compared to the motion of the same side chain in the disaccharide and in the Lewis" trisaccharide (Figure 3 ) , it was observed that transition to the minimum near 140' is more rare. This shows that the Fuc residue linked to the Gal residue, although one residue away from the GlcNAc, has some direct influence on the movement of this group. These observations are consistent with the results of two-dimensional NOESY simulations of exchangeable protons in acetamido sugars21 where a NOE contact between the NH proton and the CH3 protons of the Fuc residue was seen, and the simulated value of the torsion angle was 90" ( f20' ) . Blood Group A Tetrasaccharides with Type I and Type II Core Chains. The type I blood group A tetrasaccharide (Table I ) was studied using methods

1743

similar to those described above. The lowest energy minimum of the linkage GalNAc a- ( 1+ 3 ) Gal has been identified to be 60°, -150°.'2 In the minimized conformation of the tetrasaccharide the +,$ angles of the glycosidic linkages are -65",128", 55",-163", and -61",-114" degrees for a - ( 1 + 2)-, a-(1 + 3 ) -,and B- ( 1 + 3) linkages, respectively. This conformation was used for subsequent dynamics simulations. The time series of the individual 4,$ angles are shown in Figure 7, and the average values and the fluctuations are listed in Table 11. The trajectories and the small rms fluctuations indicate that this molecule is also relatively rigid. Modest deviations from the minimum energy conformation seen for the GalNAc a- (1 + 3)Gal linkage are shortlived and do not contribute significantly to the conformational equilibrium. The trisaccharide cores Fuc a-( 1 -P 2)Galp-( 1 + 3)GlcNAc and Fuc a-( 1 + 2) [ GalNAc a-( 1+ 3) ] Gal have been studied previously, l2 and have been found to fluctuate around a single minimum. The major conformations obtained for different linkages in these two trisaccharides have been found to be nearly the same in the tetrasaccharide also. Hence it is quite likely that this tetrasaccharide, which could be considered as a combination of these two trisaccharides, will also be quite rigid. The type I1 A tetrasaccharide (Table I ) differs from the type I A in only one linkage, with a Gal pI

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Figure 7. Trajectory of type I A core tetrasaccharide. ( a ) $ Fuc a-(1+ 2 ) , ( b ) (b Fuc a-(1 + 2 ) , ( c ) (b Gal NAca-(1 + 3 ) , ( d ) $ Gal NAca-(1 + 3 ) , ( e ) (b Gal p(1+3),and(f)$Gal@(l+3).

1744

MUKHOPADHYAY AND BUSH

( 1+ 4) linkage instead of a p- ( 1 + 3) linkage. The linear trisaccharide core Fuc a-( 1+ 2) Gal B- ( 1 4 ) GlcNAc 6-OMe, also the blood group H antigen, has been included in the present studies. The &,$ angles of Gal 8- ( 1* 4 ) GlcNAc were taken to be -60",120", from the present studies of this disaccharide. The 4,$ angles of the minimum energy conformation of this trisaccharide were -72",132" and -55",113" for the a - ( 1 + 2)-, and the 0 - ( 1 4) linkages, respectively. The dynamics trajectories of these glycosidic dihedral angles (data not shown) does not show any conformational transition. The average values and the fluctuations of the 4 and $ angles are listed in Table 11. The type I1 blood group A tetrasaccharide was generated following the same methodology. The minimum energy conformation used for the dynamics simulations has the 4,$ angles of -64",128", 54",-159", and -54",116" for the a(1+2)-,a-(l-*3)-,andP-(1+4)linkages,respectively. The time series of these angles are shown in Figure 8, and the average values and the fluctuations are listed in Table 11. This tetrasaccharide has also been found to fluctuate around a single minimum energy conformation with a very few short-lived deviations in GalNAc a- ( 1+ 3) linkage. The common glycosidic linkages present in both chains, i.e., Fuc a- (1+ 2) and GalNAc a-( 1+ 3), --f

--+

IJO 100

> 1 a

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Figure 8. Trajectory of type I1 A core tetrasaccharide. ( a ) J/ Fuc a - ( 1 + 2 ) , ( b ) 6 Fuc a - ( 1 --t 2 ) , ( c ) J/ Gal NAccw-(1 + 3 ) , ( d ) Gal NAca-(1 + 3 ) , ( e ) J. Gal p( 1 + 4 ) , and ( f ) 4 Gal 8-(1+ 4 ) .

have been found to have nearly the same values in these two tetrasaccharides. In other words, the common branched trisaccharide core Fuc a - ( 1 + 2 ) [ GalNAc a- ( 1+ 3) ] Gal preserves the same conformation in both chains and the results are very similar to a previous MD study of this trisaccharide.12 The present study shows that the 4,$ values of the p-( 1 + 3 ) - and p - ( 1 + 4)linkage, which distinguishes the type I and type I1 core chains, do not show great conformational flexibility. These angles adopt quite different values, the average values of the former linkage are -62",-112" and that of the latter are -52",117", respectively. Hence, even if the common trisaccharide core has the same conformation, the overall three-dimensional structure of these two tetrasaccharides are quite different. Although solvent molecules were not included explicitly in our calculations, the major conformations obtained from the simulations are very similar to those obtained from the NOE experiments carried out on oligosaccharide alditols R5 and R9, isolated from ovarian cyst mucin glycoproteins, each carrying the blood group A determinant on type I1 and type I cores, respectively,22however, the 4 angle of the Gal 0- ( 1 + 4)linkage in the type I1 A tetrasaccharide obtained from the present simulations differs by x 3 0 " from that obtained from the NOE experiments ( Table TI ) .

DISCUSSION MD simulations involve the generation of a representative conformational space of any molecule. When applied to carbohydrates it has indeed provided a wealth of information regarding the various motions occurring in the molecule. The chair conformation of the pyranose ring undergoes numerous fluctuations on a subpicosecond time scale, which gives rise to small puckering in the ring. The disaccharides undergo conformational transitions, brought about mainly by the changes in the glycosidic dihedral angles. The side chains also undergo transitions between different minima. However, for the case of the blood group oligosaccharides in this study, extension of disaccharides to tri- and tetrasaccharides introduces more stringent steric restrictions and as a result the oligosaccharidesmainly fluctuate around a single conformational energy minimum with deviations on the order of 10" in the glycosidic dihedral angles. It is well known that the choice of starting conformations can limit the conformational space sampled by a molecule during the dynamics simulations.

MD OF LEWIS BLOOD GROUPS

In order to avoid that, multiple trajectories were simulated for the same molecule each with different set of initial velocities and several hundred picoseconds of dynamics was recorded for each of the molecule. The average rms fluctuation of the potential energy of these molecules obtained during the simulation period was about 2-4 kcal/mol at room temperature. Hence it is likely that the numerous thermal fluctuations that take place at this temperature will sample any existing minimum separated from the starting conformation by a barrier less than a few kilocalories during these long simulations. The lack of spontaneous conformational transitions to other minima during the simulations suggests that these oligosaccharides can maintain well-defined conformations with relatively long lifetimes. The disaccharide energy maps (Figure 1) are also quite restricted, having only a very few energy minima due to nonbonded repulsions. In forming the triand tetrasaccharides, some of the disaccharide conformations are eliminated by additional nonbonded interactions. We doubt that any low-energy minima have been overlooked in the highly restricted energy space available to these relatively small molecules. It should not be too surprising that the blood group oligosaccharides included in this study were found to be relatively rigid. The conformational properties of one of them can be extrapolated from those of closely related structures previously studied by our research group. The trisaccharide cores Fuc a- ( 1 --* 2 ) Gal p- ( 1 + 3 ) GlcNAc and Fuc a- ( 1 -+ 2 ) [ GalNAc a- (1+ 3 ) Gal have been studied previously,12and both were found to fluctuate around a single minimum energy conformation. Thus the conformation and dynamics of the type I blood group A tetrasaccharide studied in this work could be considered to be a fusion of the two previously studied trisaccharides. Indeed our present results are consistent with this interpretation. Similarly, the Lewisb oligosaccharide can be considered to be a fusion of the Lewisd trisaccharide, Fuc a- (1 + 2)Gal p( 1 + 3 ) GlcNAc, and the Lewis" trisaccharide, both of which adopt relatively rigid conformations. Thus models of the Lewisb tetrasaccharide show it to be extremely compact and almost spherical in shape.g Interestingly, the conformation observed for the Fuc a - (1 + 4)GlcNAc linkage in the major conformation of the Lewis" and Lewis oligosaccharides is found to be a minor conformation not at the minimum energy in the isolated disaccharide. We have been able to identify steric interactions between the Gal and the Fuc residues linked to the GlcNAc in the Lewis" and Lewisboligosaccharides, and this interaction has been experimentally observed in

1745

NOESY cross peaks between Gal H2 and Fuc H5 in studies on LNF-2 and Lacto-N-difucohexaose-1 (LND-l).' The orientation of the amide in 2-deoxy-2-acetamido sugars has been previously addressed by measurements of CD spectra and of the amide proton coupling c o n ~ t a n t . *Values ~ ' ~ ~ of 3JcHin the range of 8-10 Hz have been interpreted as an indication of a nearly trans arrangement of the amide proton and H2. However, conformational calculations such as those reported in this work indicate that the orientation should not be exactly trans ( 7 = 120") but slightly displaced so that 7 = 100" or 140". Our simulation of Fuc a- ( 1-P 4 ) GlcNAc implies that both conformations are populated, but for the Lewis" and Lewisb models, the form with T = 100" predominates. Thus, the behavior of T is somewhat similar to that of the glycosidic dihedral angles $C and $; steric interactions in the blood group oligosaccharide limit the range of motion of the amide side chain. Experimental evidence on the values of 7 in blood group oligosaccharides is somewhat limited. The amide proton coupling constant is not very sensitive to 7 in this range, but NOESY cross peaks of the amide proton have been reported that are more sensitive to the amide orientation. The data indicate that 7 values near 90" are preferred in LND-1 and in LNF-1, and that there seems to be a subtle difference in the orientation of the amide plane in these milk oligosaccharides. CDs in the 210-nm region, which is also sensitive to the amide plane orientation, suggest that the amide orientation may differ in LND-1 and LNF-1." Our MD simulations on LNF-2 and LND-1 show how the interaction of the FUC'in the latter compound with the amide group may produce the changes in amide dihedral angle. It is instructive to compare the results of our simulations with oligosaccharide conformations deduced from 'H-nmr data interpreted by NOESY simulations. The comparison between theoretically simulated conformations with the solution conformations obtained from nmr experiments shows generally good agreement9'21'22 (see Table 11). The observed NOES indicated that these molecules can adopt a narrowly defined conformational region in solution. The estimated experimental error in the glycosidic dihedral angles determined from the NOESY data are approximately the same as the subpicosecond fluctuations of these same angles in our MD simulations. The only case in which there is obvious disagreement between the conformations deduced from NOESY simulations and from these MD simulations is the case of the Gal p-(1 +

1746

MUKHOPADHYAY AND BUSH

4)GlcNAc linkage in the type I1 A blood group oligosaccharide, for which the dihedral 4 differs by about 30’ (Table 11). Seeking possible sources of

This research was supported by NIH grant GM-31449.

REFERENCES this discrepancy, one must consider the inadequacy 1. Watkins, W. M. ( 1972) in The Glycoproteins, 2nd ed., of the potential functions. While it is difficult to part B, Gottschalk, A., Ed., Elsevier, Amsterdam, pp. evaluate the numerous possible criticisms of the 841. Rasmussen potentials, it seems unlikely that this 2. Kabat, E. K. (1982) Am. J . Clin. Pathol. 87, 281discrepancy of 30’ could be resolved by any modi292. fications in the potential functions. The most ob3. Feizi, T. (1985) Nature 314,53-57. vious weakness of the NOESY simulation method 4. Brandley, B. K., Swiedler, S. J. & Robbins, P. W. (1990) Cell 63,861-863. is the assumption of a rigid isotropic tumbling model 5. Carver, J. P., Michnick, S. W., Imberty, A. & Cumfor the oligosaccharide. This model neglects the efming, D. A. (1989) in Glycoprotein and Biological Recfects of internal motion and does not correctly acognition, CIBA Foundation Symposium, Harnett, S., count for conformational a ~ e r a g i n gHowever, .~ the Ed., John Wiley & Sons, London. MD simulation supports the assumption that the 6. Scarsdale, J. N., Prestegard, J. H. & Yu, R. K. (1990) Gal p-( 1 + 4 ) GlcNAc linkage is relatively rigid. Biochemistry 29,9843-9855. The discrepancy arises from the small difference in 7 Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S. the conformation predicted by the NOESY and MD & Rao, V. S. (1980) Can. J . Chem. 58,631-653. simulations. Lastly, one must consider the possi8. Yan, Z.-Y., Rao, B. N. N. & Bush, C. A. (1987) J . bility of some experimental error in the NOESY data Am. Chem. SOC.109, 7663-7669. that has escaped our attention. This particular link9. Cagas, P. & Bush, C. A. (1990) Biopolymers 30,11231138. age has not received much experimental attention, 10. Bush, C. A. & Cagas, P. (1991) in Advances in Bioand there are some technical problems caused by physical Chemistry, Vol2, Bush, C. A., Ed., JAI Press, unfortunate overlaps in the chemical shifts of Greenwich, CT, in press. GlcNAc H2, H3, and H4 that complicate extraction 11. Brady, J. W. (1990) Adu. Biophys. Chemistry, Vol. 1, of accurate NOE values. It is possible that new Bush, C. A., Ed., JAI Press, Greenwich, CT, pp. 155methods of heteronuclear nmr with 13C resolution 202, and references therein. of the chemical shifts might provide some valuable 12. Yan, Z.-Y. & Bush, C. A. (1990) Bwpolymers 29,799new experimental information on the conformation 811. of the Gal p- ( 1 + 4 ) GlcNAc linkage. 13. Verlet, L. (1967) Phys. Rev. 159,98-103. While the solvent is not explicitly treated in these 14. van Gunsteren, W. F. & Berendsen, H. J. C. (1977) simulations, neither are they true vacuum MD simMol. Phys. 34, 1311-1327. 15. Rasmussen, K. (1982) Acta Chem. Scand. Ser. A 36, ulations since the potential functions are fit to con323-327, and references therein. densed phase experimental data.” The experimental 16. Ha, S. N., Giammona, A., Field, M. & Brady, J. W. nmr data with which our results are compared are (1988) Carbohydr. Res. 180,207-221. all from studies in solution, usually aqueous. Our 17. Brady, J. W. (1986) J . Am. Chem. SOC.108, 8153simulations, in agreement with earlier modeling 8160. calculations, 7,8 show that nonbonded interactions 18. Brady, J. W. (1987) Carbohydr. Res. 165,306-312. predominate with little evidence that hydrogen 19. Prabhakaran, M. & Harvey, S. C. (1987) Biopolymers bonding or solvophobic forces are important in de26,1087-1096. termining these conformations. Therefore, one pre20. Rao, B. N. N., Dua, V. K. & Bush, C. A. (1985) Biodicts that the conformation of the blood group olipolymers 2 4 , 2207-2229. gosaccharides should not depend strongly on the 21. Cagas, P., Kaluarachchi, K. & Bush, C. A. (1991) J . temperature or solvent conditions. NOE s t u d i e ~ ~ ’ ~ Am. Chem. SOC.113, 6815-6822. 22. Cagas, P. & Bush, C. A. (1991) Biopolymers (in press). in MezSO and in pyridine solution have provided 23. Bush, C. A., Duben, A. J. & Ralapati, S. (1980) Bioexperimental support for this conclusion. Therefore chemistry 19, 501. we claim that simulations that neglect explicit ef24. Homans, S. W. (1990) Biochemistry 29,9110-9118. fects of solvent may be more relevant to solution 25. Edge, C. J., Singh, U. C., Bazzo, R., Taylor, G. L., conformations than would be suggested by simple Dwek, R. A. & Rademacher, T. W. (1990) Biochemconsiderations. Although the precise time scale of istry 29, 1971-1974. the conformational fluctuations may be influenced by solvent, as suggested by Homans (1990)24and Edge et al.,” we believe the present simulations give Received June 24, 1991 Accepted September 16, 1991 a good account of their type and magnitude. I .