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Conformations of terminal sialyloligosaccharide fragments — a molecular dynamics study
Journal of Theoretical Biology 222 (2003) 389–402
Conformations of terminal sialyloligosaccharide fragments — a molecular dynamics study M. Xavier Suresh, K. Veluraja* Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627 012, India Received 30 May 2002; received in revised form 14 November 2002; accepted 31 December 2002
Abstract Molecular dynamics simulations have been performed to understand the conformational features of the terminal sialyloligosaccharide fragments NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc. The conformational regions Ai ; Bi and Ci were identified in the Ramachandran plot. Analysis of the 1000 ps trajectories collected through simulation (2000 ps in the case of NeuNAca (2-9)NeuNAc) revealed that these molecules have conformational propensity in region Bi. The occurrence of these molecules in the common conformational space leads to a structural similarity between them. This structural similarity may be an essential requirement for the neuraminidase activity towards sialyloligosaccharides. The local change in the conformation of the active site residues of neuraminidases may contribute for the specificity differences between different linkages of sialyloligosaccharides. A highly conserved water-mediated hydrogen bond observed in these structures between the sugar residues, acts as an additional stabilizing force. r 2003 Published by Elsevier Science Ltd. Keywords: Sialyloligosaccharide; Molecular dynamics; Structural similarity
1. Introduction Cell surface carbohydrates deriving from glycolipids, glycoproteins and proteoglycans mediate the tight associative interactions between molecules and cells or between cells and cells at the origin of numerous recognition processes (Varki, 1993; Dwek, 1996). The role of carbohydrates in biological recognition is known to be of great importance, not now but for quite a long time. The first indication of such a role came from the discovery by Hirst (1942a, b) that the influenza virus agglutinates erythrocytes. It was later shown by Gottschalk (1957) that this virus binds to the red cells through sialic acid residues present on the cell surface. Cell surface carbohydrates are responsible for such phenomena as infectious interaction, embryonic development, blood group specificity and clearance of senescent cells (Hughes, 1976; Jeanloz and Codington, 1976; Sharon, 1979; Holmgren et al., 1980).
*Corresponding author. Tel.: +1-91-462-333887; fax: +1-91-462322973. E-mail address: [email protected] (K. Veluraja). 0022-5193/03/$ - see front matter r 2003 Published by Elsevier Science Ltd. doi:10.1016/S0022-5193(03)00050-X
Sialic acid is an unusual acidic amino sugar present in glycolipids, glycoproteins, oligosaccharides and polysaccharides. It is one of the important building blocks of gangliosides and play a vital role in a variety of fundamental physiologically and pathologically important processes (Rosenberg, 1995; von Itzstein and Colman, 1996; Reutter et al., 1997; Mammen et al., 1998). Numerous derivatives of sialic acids have been identified (Kelm and Schauer, 1997; Schauer et al., 1997), of which N-acetylneuraminic acid (NeuNAc) is the most abundant one. Very small quantities of sialic acids are also found in a free state. Though sialic acid occurs as the a-anomer in gangliosides, the b-anomer is predominant in solution; the b-anomer has also been found in the crystalline state (Flippen, 1973; Bhattacharjee et al., 1975). They occur terminally or in buried form in sialyloligosaccharides. An analysis across the CCSD, a database for complex carbohydrate structures, shows that the sialic acid predominantly occurs as a terminal sugar in the complex carbohydrate structures (Doubet et al., 1989; Christlet et al., 2000). The terminal sialic acid is attached to the penultimate sugar with specific type of linkages. The predominant sialic acid linkages that are prevalent in sialyloligosaccharides are:
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NeuNAca(2-3)Gal–R; NeuNAca(2-3)GalNAc–R; NeuNAca(2-6)Gal–R; NeuNAca(2-6)GalNAc–R; NeuNAca(2-8)NeuNAc–R and NeuNAca(2-9)NeuNAc–R (Varki et al., 1999), where the R indicates the penultimate bulky chains of the specific type of glycosidic linkage. Studies indicate that the sialyloligosaccharide fragments are the substrates for many enzymes such as neuraminidases (Corfield et al., 1981, 1983; Suzuki et al., 1986; Baker et al., 1987; Daniels et al., 1987; Kopitz et al., 1996; Gornati et al., 1997) that cleave terminal sialic acids. Influenza virus neuraminidase can accommodate a minimum of two residues alpha ketosidically linked with sialic acid (Veluraja et al., 2001). And Endoneuraminidase N, derived from the Escherichia coli K1-specific bacteriophage PK1, requires a minimum of eight a2,8-linked sialic acids for binding and cleaves poly sialic acid into oliogomers of two to seven residues (Finne and Makela, 1985; Hallenbeck et al., 1987). They are the receptor site for many biological macromolecules and are involved in various life processes. The extreme structural diversity possible for oligosaccharides is the key to their postulated biological roles. Carbohydrate structural and conformational analysis is a prerequisite for further studies involving carbohydrate–protein interactions which are important, e.g., in protein trafficking, inflammation or bacterial infection processes. The analysis may be divided into the analysis of the primary structure as well as the three-dimensional structure. The former deals with the identity of the constituent monosaccharides, substituents if any, and the sequence of the monosaccharide residues and the latter includes conformation, flexibility and dynamics which are important properties to investigate in order to understand the function of carbohydrates in relation to other molecules, most often proteins. It is obvious that a detailed knowledge of the structure and dynamics of carbohydrates, both free and bound to proteins is indeed relevant (Bush et al., 1999). In order to gain information about the probable conformation and dynamics of the oligosaccharides, the molecular dynamics technique has been used. It can also assist in the interpretation of NMR data, in the study of conformational flexibilities, in the analysis of the conformational variability of the glycosidic linkages, etc., other than the efficient searches of low-energy structures in the case of complex carbohydrates. As sialic acid is the sugar found at the non-reducing end of glycolipids and glycoproteins, and is a cleavage product of the sialyloligosaccharides by the sialidases or neuraminidases, investigation of the conformational dynamics of these sialyloligosaccharide fragments is essential to understand its molecular recognition. The conformation of oligosaccharides and polysaccharides containing the disaccharides moieties NeuNAca(23)Gal, NeuNAca(2-6)Gal or NeuNAca(2-8)NeuNAc
as their part have been studied in the past by various other groups using NMR spectroscopy and computational methods (Breg et al., 1989; Blanco et al., 2000; Acquotti et al., 1990; Poppe et al., 1989; Baumann et al., 1993; Yamasaki and Bacon, 1991; Veluraja and Rao, 1983; Mukhopadhyay and Bush, 1994; Vasudevan and Balaji, 2002). The present work aims at having a complete picture about the conformational preferences and flexibility of the four disaccharide fragments, NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(28)NeuNAc and NeuNAca(2-9)NeuNAc that are prevalently observed in sialyloligosaccharides using molecular dynamics simulations over a period of 1000 ps with the explicit inclusion of water molecules. 2. Materials and methods 2.1. Initial model In the present study, the disaccharide fragments of sialyloligosaccharides that have been considered are NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(28)NeuNAc and NeuNAca(2-9)NeuNAc and their structures are depicted in Fig. 1. The internal parameters such as bond length, bond angle and torsion angle which were obtained from the standard geometry in 2C5 chair form (Flippen, 1973; Veluraja and Rao, 1980) were used for the generation of the coordinates for the sialic acid and hence the sialyloligosaccharide fragments. 2.2. Construction of glycosidic linkages to form sialyloligosaccharide fragments While the construction of glycosidic linkages to form sialyloligosaccharide fragments, the net effect was the removal of a molecule of water upon forming the glycosidic linkage. It is necessary to take care of the allowed conformational flexibility of the disaccharide fragments, which arises due to the freedom of rotation around glycosidic torsions and the exocyclic bond. The definitions for these dihedral angles (Fig. 1) are as follows: (a) NeuNAca(2-3)Gal: fg ¼ C1 C2 O2 C3; cg ¼ C2 O2 C3 H3: (b) NeuNAca(2-6)Gal: fg ¼ C1 C2 O2 C6; cg ¼ C2 O2 C6 H61; w ¼ O2 C6 C5 O5 (H61 is that hydrogen which makes an angle of H61–C6–C5–O5=120 when w=0 )
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Fig. 1. The structure of terminal sialyloligosaccharides together with the torsions labeled: (a) NeuNAca(2-3)Gal, (b) NeuNAca(2-6)Gal, (c) NeuNAca(2-8)NeuNAc, and (d) NeuNAca(2-9)NeuNAc.
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(c) NeuNAca(2-8)NeuNAc: fg ¼ C1 C2 O2 C8; cg ¼ C2 O2 C8 H8; w2 ¼ C9 C8 C7 C6; w1 ¼ C8 C7 C6 C5 (d) NeuNAca(2-9)NeuNAc : fg ¼ C1 C2 O2 C9; cg ¼ C2 O2 C9 H91; w3 ¼ O2 C9 C8 C7; w2 ¼ C9 C8 C7 C6; w1 ¼ C8 C7 C6 C5 (H91 is that hydrogen which makes an angle of H91-C9-C8-C7=120 when w3 =0 ). The glycosidic angle is fixed at 117.5. The carboxylic acid group of sialic acid is considered to be ionized in neutral pH and hence the total charge per sialic acid is 1. The charge assigned to the atoms around the carboxylic acid group of sialic acid are C2=0.379 and C1=0.679. The charge associated with each of the oxygen atoms of C1 is 0.792. No counter ion is included. All the hydrogen atoms were treated explicitly and all-atom simulations have been performed.
The water molecules were added from the library of the modeling software Insight II. The numbers of water molecules differ from structure to structure. The resulting ensemble was energy minimized using steepest descent and conjugate gradient methods until the ( 1. The potential gradient reduces to 0.01 kcal mol1A temperature of the system is maintained at 300 K. The width of the integration steps of the MD simulations was 1fs at a 64-bit precision of the computer word. Non-bonded interactions were calculated on the basis of ( under periodic atom pair with a cut-off radius 20 A boundary conditions. The history of information was recorded for every 500 steps of trajectory. The whole system is subjected to MD simulations for a period of 1000 ps were performed followed by a 30 ps equilibration except for NeuNAca(2-9)NeuNAc. The simulation period is extended to 2000 ps for NeuNAca(2-9)NeuNAc considering the flexibilities of the exocyclic torsions. To examine the structural and conformational variations during these simulations, structures from the respective local minima were selected systematically. And the selected structures were further energy minimized using steepest descent method for 100 cycles with a subsequent conjugate gradient method until the energy ( 1. The MD gradient dropped below 0.01 kcal mol1 A trajectory informations were analyzed using ANALYSIS module of Biosym package.
2.3. Computational procedures 3. Results and discussion The molecular modeling studies were carried out on a Silicon Graphics IRIS Crimson Elan graphics workstation using Biosym, a model building package by Molecular Simulations Inc. (MSI). Insight II was the graphical interface. Molecular mechanics calculations were carried out in the AMBER force field which incorporates carbohydrate force field (Weiner et al., 1984; Weiner et al., 1986; Homans, 1990) using DISCOVER module (an energy minimization program) by MSI. The distance-dependent dielectric constant e ¼ 4rij was used. When simulating a microscopic system of finite size, the boundary of the system should be treated such as to minimize edge effects the standard procedure is to use periodic boundary conditions. The biomolecule and its surrounding solvent molecules are put into a periodic space-filling box, which is treated as if it is surrounded by its identical translated images of itself. In ( is this present study, a box of dimension 20 20 20 A constructed for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal and NeuNAca(2-8)NeuNAc and the solvated sialyloligosaccharide fragments are placed at the center. For the disaccharide NeuNAca(2-9)NeuNAc which is likely to have larger extension, hence the box size of ( was considered. This can facilitate the 25 25 25 A free movement of the molecule during the dynamics.
In our attempt for the detailed conformational analysis of the solvated carbohydrates, especially of terminal disaccharide fragments of sialyloligosaccharides, we performed a series of different extended MD simulations. The incorporation of water in the simulations is mandatory for a reasonable description of conformations and entropy. Raising the temperature distorts the ring geometry of sialic acid, and hence the simulations with temperature 300 K were considered for further analysis. 3.1. Disaccharide NeuNAca(2-3)Gal Molecular dynamics was carried out for the disaccharide NeuNAca(2-3)Gal with explicit water over a period of 1000 ps with a history of structures at every 0.5 ps which leads to a total of 2000 structures. Fig. 2 shows the distribution of glycosidic torsions in the (fg ; cg ) space for the disaccharide, from this simulation. From the figure it is clear that there are two distinct regions in the conformational space indicated as region A1 and region B1. A total of 100 structures selected from these regions were energy minimized, only the relative energies of global and local minimum conformations are
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Fig. 2. The (fg ; cg ) distribution in NeuNAca(2-3)Gal. The allowed regions are noted as A1 and B1. Table 1 Minimum energy conformers for NeuNAca(2–3)Gal No.
f (deg)
c (deg)
Relative energy (kcal mol1)
1 2
66 164
10 59
0.0 3.0
given in Table 1. It is clear from the table that region B1 is the global minimum energy region and region A1 is a local minimum energy region and the energy difference being 3 kcal mol1. In the earlier work using the hard sphere energy calculations, Veluraja and Rao (1983, 1984) suggested that there are three conformational regions possible for this NeuNAca(2-3)Gal linkage. In another work, Mukhopadhyay and Bush (1994) reported four local minimum regions. However, the global minimum conformations observed in these studies are also around the region that is same as that of the region B1 as in the present study and the other high-energy regions are not observed in this present study. It is also to be mentioned here that the terminal NeuNAca(2-3)Gal in ganglioside GD1a also favor the conformational region B1 (Veluraja and Rao, 1983). Earlier work on the conformational aspects of GM4 indicates that there are three allowed conformational regions accessible for the same fragment NeuNAca(2-3)Gal with the minimum in region A1 (Poppe et al., 1989). In the same study, the NOE experimental data showed that the population is more (about 60%) in region B1. For the NeuNAca(2-3)Gal fragment in GM1, Acquotti et al. (1990) were able to get a conformation in region A1 using NOE distance– mapping procedure. Similar conformational preference is also observed in the earlier hard sphere energy
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calculations (Veluraja and Rao, 1983). However, this conformational preference has already been accounted for the vicinal location of the GalNAc residue attached to the galactose residue. Hence, the fragment in GM1 cannot be equated to the terminal fragments. In the present molecular dynamics study, of the two conformational regions accessible, the region B1 is more populated as evidenced from the dynamics trajectory which is in accordance with the NOE data of Poppe et al. (1989). Even though various starting conformations for the molecule from the other regions given, the molecule tend to be in the region B1 and the transitions are observed only between the A1 and B1 more frequently. A representation plot from the dynamics trajectory clearly shows the transitions between regions A1 and B1 (Fig. 3a). The starting conformation for this plot is from region B1. The dynamics has been further extended over a period of 3000 ps and no such transitions to the regions other than A1 and B1 have been observed (data not shown). The absence of accessibility of the conformational space in the third region (as identified by hard sphere energy calculations) other than A1 and B1 may be due to the high-energy barrier separating these regions. The global minimum energy conformation of NeuNAca(2-3)Gal is well stabilized by water-mediated hydrogen bonds (Fig. 4). The significance of the watermediated hydrogen bonding interactions in the global minimum conformational space has been brought into light by an exhaustive analysis over the complete 1000 ps dynamics trajectory. It reveals that in more than 50% of the structures, a molecule of water is invariably present at a particular position and do mediate a hydrogen bond interaction between O7 of NeuNAc and O2 of Gal, whenever this disaccharide assumes the conformations in the global minimum energy region B1. Even though different water occupies that particular position, the position is highly conserved. 3.2. Disaccharide NeuNAca(2-6)Gal Analysis of the MD trajectory records of the simulated structures of NeuNAca(2-6)Gal reveals interesting features. Fig. 5 depicts the dynamics behavior of the glycosidic torsions of NeuNAca(2-6)Gal in solvent environment over a period of 1000 ps molecular dynamics run. From the figure it is inferred that there are two conformational regions available, region A2 and B2. The regions are roughly similar to the one that has been found for the disaccharide NeuNAca(2-3)Gal. The relative energies for the different conformational regions were worked out by systematically sampling the structures from the trajectory records as mentioned earlier (100 structures). The possible conformations and their relative energies are given in Table 2. If we have
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Fig. 3. (a-d) The representative plots of MD trajectory showing the transitions of the glycosidic torsions fi ; ci for the disaccharide fragments.
a closer look into Table 2, it is noticed that in the minimum energy conformation the glycosidic torsion (fg ; cg ) favor values around (52 , 88 ) which is in the region B2. An energy difference of 13 kcal mol1 is noticed between region A2 and region B2. And hence it is clear from the table that the conformational propensity for these molecules is in region B2.
It is interesting to observe, in this disaccharide NeuNAca(2-6)Gal, the behavior of the exocyclic torsion (w), since it provides an additional freedom of rotation and hence enhances the flexibility of the molecule. From Table 2 it is found that the exocyclic torsion w for the minimum energy conformation is 73 . But it can also assume other possible staggered values (60 and 180 )
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Fig. 4. The global minimum energy conformation of NeuNAca(2-3)Gal with the typical water-mediated interaction.
population with different w values, w ¼ 60 ; 180 , 60 ; are 83%, 4%, 13%, respectively, gives a clear indication of the predominance in +60 conformation. Analysis of the structures in the global minimum energy conformational space indicates that a watermediated hydrogen bonding interaction is formed either between O7 of the first residue and O5 of the second residue (Gal) or between O8 of the first residue and O4 of the second residue. However, the former is more prevalent than the later. A representative structure in the global minimum energy conformation with watermediated hydrogen bonding interaction is shown in Fig. 6. This water approximately occupies the same location in space as in the case of NeuNAca(2-3)Gal. 3.3. Disaccharide NeuNAca(2-8)NeuNAc
Fig. 5. The (fg ; cg ) distribution in NeuNAca(2-6)Gal. The allowed regions are noted as A2 and B2.
Table 2 Minimum energy conformers for NeuNAca(2–6)Gal No.
f (deg)
c (deg)
w (deg)
Relative energy (kcal mol1)
1 2 3 4 5
52 60 178 69 179
88 74 55 36 51
73 57 170 172 68
0 8.5 13.4 14.3 22.3
with possibility of an increase in energy (Table 2). For example, a difference of 8.5 kcal mol1 is accounted when a switching occurs from values near +73 to values around 57. It is important to note that in the earlier hard sphere energy calculations the favored value for w is also around 73 . An analysis of the available
The molecular dynamics calculations that reveal the glycosidic conformational flexibility of NeuNAca(28)NeuNAc is shown in Fig. 7. From the figure it is clear that the molecule can be in two regions, region A3 and region B3. The conformation of the molecule occurred for longer duration is in region B3. Minimization of the selected structures reveals that the global minimum is in the region B3 (Table 3). The glycosidic torsion along with the exocyclic rotations involved in this linkage (fg ; cg ; w2 ; w1 ) prefers values around (63 , 5 , 60 , 169 ) in the global minimum energy conformation. These are in agreement with the conformations deduced earlier based on energy calculations (Veluraja and Rao, 1984). A difference of 6 kcal mol1 is noticed between the region A3 (local minimum) and region B3 (global minimum). Recently, Vasudevan and Balaji (2002) explored the conformational details of the disialosyl fragment NeuNAca(2-8)NeuNAc using molecular dynamics for a period of 1 ns with another force field, GROMOS96 (Gunsteren and Berendson, 1996). As evidenced from
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Fig. 6. The global minimum energy conformation of NeuNAca(2-6)Gal with the typical water-mediated interaction.
hydrogen bonding interactions. Carefully analyzing the water-mediated interactions, it is interesting to note that there is a water molecule that has been present at a hydrogen bonding distance from O7 of the first residue is being interacting with O9 of the second residue. That is, it has been involved in the water-mediated hydrogen bonding interaction between the two sugar residues and thereby stabilizing the structure of the disaccharide. The global minimum energy conformation with watermediated hydrogen bonding interaction is depicted in Fig. 8. 3.4. Molecular dynamics of disaccharide NeuNAca (2-9)NeuNAc
Fig. 7. The (fg ; cg ) distribution in NeuNAca(2-8)NeuNAc. The allowed regions are noted as A3 and B3.
Table 3 Minimum energy conformers for NeuNAca(2-8)NeuNAc No. f (deg) c (deg) w2 (deg) w1 (deg) Relative energy (kcal mol1) 1 2 3 4
63 163 47 56
5 17 16 21
60 93 58 92
169 176 65 176
0.0 5.8 7.8 9.1
earlier studies that the use of the different force fields do not significantly affect the overall conformation of the oligosaccharides despite little variations (Qasba et al., 1994; Woods, 1998; Vasudevan and Balaji, 2002). The structures in minimum energy conformations are stabilized largely by inter residue water-mediated
The 2000 ps molecular dynamics simulation of NeuNAca(2-9)NeuNAc reveals that the glycosidic conformational angle of this disaccharide fragment prefers three distinct conformational space in the (fg ; cg ) Ramachandran plot which are designated as regions A4, B4 and C4 (Fig. 9). The energy minimization results of the 200 systematically selected structures (collected for every 10 ps) are shown in Table 4. It is obvious from the table that in the global minimum, the glycosidic conformation occurs in the allowed region B4. However, when the conformation is shifted to the allowed regions A4 and C4, the energy of the molecule increases to 5.8 kcal mol1 (conformer 3) and 12.0 kcal mol1 (conformer 8). The high-energy region C4 is accessible only for a very little period in the dynamics trajectory. The conformational preference of the exocyclic torsional angle involved in the linkage reveals that w1 tends to favor a trans conformation (w1 E171 ). In the minimum energy conformations, w2 and w3 favor values around 180 , 760 . An inspection of the time plots of w2 and w3 (Fig. 10a and b) reveals that all the three staggered conformations are accessible for these two torsions however, in the major period of simulated time w2 favors
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Fig. 8. The global minimum energy conformation of NeuNAca(2-8) NeuNAc with the typical water-mediated interaction.
Fig. 9. The (fg ; cg ) distribution in NeuNAca(2-9)NeuNAc. The allowed regions are noted as A4, B4 and C4.
a value of 60 and w3 favors a value of +60 and 180 . w2 can also favor a value of +60 and w3 ; 60 with a lesser period of time in the simulation which is also reflected in the minimum energy conformations. When w2 and w3 prefer values around 60 , the relative energy of the system is 5.8 kcal mol1 and when w2 and w3 prefer values around +60 , the relative energy is 5.9 kcal mol1. However, when w1, w2 and w3 takes up a trans conformation (wi’s =180 ), the conformational energy increases to 26.1 kcal mol1 (conformer 10).
In the global minimum energy conformation direct and water-mediated hydrogen bonding plays a predominant role in the stabilization of the structure (Fig. 11). In this conformation a direct hydrogen bonding interaction is observed between O9 of the first sialic acid and the acetamido carbonyl of the second sialic acid and between O8 of the first sialic acid and carboxyl of the carboxylic acid group of the second sialic acid. A water also bridges the disialic acid by mediating a hydrogen bond between O7 of first sialic acid and the carbonyl of the carboxylic acid of second sialic acid. When the torsional angle w3 in the global minimum conformation is shifted from 173 to 98 (conformer 2, energy difference 4.5 kcal mol1), the hydrogen bonding pattern changes completely. In this conformation only a single hydrogen bond is possible. The hydrogen bond can be either a direct, between O7 of the first sialic acid and the N5 of the second sialic acid or a water mediated hydrogen bond between these two atoms. The loss of two hydrogen bonds perhaps accounts for this energy difference. The structures in other conformations have also been stabilized by hydrogen bonding interactions. An earlier investigation based on molecular modeling of this substrate into the active site of influenza virus N9 neuraminidase (Veluraja et al., 2001) revealed that the conformational parameter that the enzyme can accommodate at its binding pocket are (fg ; cg ; w1 ; w2 ; w3 )= (73, 49, 165, 176, 162). A conformational structure close to the above parameter is observed in our molecular dynamics calculation (conformer 10), but the energy of the conformer is about 26.1 kcal mol1 higher than that of the global minimum. However, this conformer differs from the global minimum energy
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Table 4 Minimum energy conformers for NeuNAca(2-9)NeuNAc No.
f (deg)
c (deg)
w3 (deg)
w2 (deg)
w1 (deg)
Relative Energy (kcal mol1)
1 2 3 4 5 6 7 8 9 10
67 45 175 78 173 68 63 64 62 58
68 76 47 62 45 44 65 72 64 66
173 98 61 49 168 50 60 54 59 175
62 57 53 53 56 61 176 92 175 170
174 179 170 157 174 165 179 163 176 176
0.0 4.5 5.8 5.9 6.8 7.7 7.8 12.0 23.3 26.1
Fig. 10. The time plot showing (a) the preference of w2 and (b) the preference of w3 :
conformer only in the favored value of w2 : Hence, there is a possibility that the enzymes may take the global minimum energy structure in the binding pocket and subsequently shift the conformation of w2 value from 170 to 60 before cleaving it.
3.5. Comparison of the conformations of disaccharide fragments An in-depth analysis of the conformational dynamics of the disaccharides reveals that the distribution of the
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Fig. 11. The global minimum energy conformation of NeuNAca(2-9)NeuNAc showing the direct and water-mediated hydrogen bonding interactions.
glycosidic conformations (fg ; cg ) of the disaccharide fragments of sialyloligosaccharide NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc, are roughly similar. It is clear from the molecular dynamics of the (fg ; cg ) plots, the disaccharide fragments of sialyloligosaccharides shares a common conformational space for glycosidic torsion irrespective of the type of linkages. The distributions have been restricted mainly into two allowed regions Ai and Bi (i=1–4) in the Ramachandran plane and an additional region C4 for NeuNAca(2-9)NeuNAc, which is a high-energy region (12.0 kcal mol1) and is accessible for a relatively smaller duration. For these disaccharides the global minimum has been observed in the region Bi. The local minima occur in region Ai, but the energy difference between the two regions for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca (2-8)NeuNAc and NeuNAca(2-9)NeuNAc is about 3.0, 13.4, 5.8 kcal mol1, and 5.8 kcal mol1, respectively. However, these molecules needs to cross barriers of 15, 33, 20 and 20 kcal mol1, respectively, to have transition from region Bi to region Ai. The transitions between these regions are found to be non-periodical and a representative plots of MD trajectory for the glycosidic torsions fi ; ci for the disaccharide fragments are shown in Figs. 3a–d. When computation was carried out using the other force field parameter (CVFF consistent valence force field) there is a shift in the magnitude of relative energy difference to the order of +1.5 kcal mol1 between the global minimum and local minima when compared with AMBER force field parameters and may not influence the conformational preference.
Further analysis on the favored conformations of these disaccharide fragments as a function of glycosidic torsion and exocyclic bond reveals a structural similarity and is schematically shown in Fig. 12. The conformers corresponds to this structural similarity are in global minimum except for of NeuNAca(2-9)NeuNAc (conformer 10). Water-mediated interactions that stabilize the structures also maintains the structural similarity. This type of structural similarity due to conformational dynamics plays a significant role in neuraminidase specificity as proposed earlier (Veluraja and Rao, 1984; Suresh and Veluraja, 2000; Veluraja et al., 2001). The finding by Kobasa et al. (1999) suggests that the adaptation of neuraminidase to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of influenza virus A neuraminidase in and around the active site to facilitate the binding of different species of sialic acid. For example, neuraminidase of N2 human influenza viruses isoleucine at position 275 is associated with high activity for NeuNAca(2-3)Gal and very low activity for NeuNAca(2-6)Gal. A change to valine results in higher NeuNAca(2-6)Gal activity without affecting NeuNAca(2-3)Gal activity. Even though the change in the conformations of the active site residues greatly alter the specificity difference of various substrates, the structural similarity in the conformations of the sialyloligosaccharides likely to play a vital role in cleavage. When these disaccharide structures favors the structural similarity, the water at a certain location has been observed to have an interaction with O7 of the first sialic acid as well as with the appropriate electronegative
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Fig. 12. The schematic representation of structural similarity observed among the disaccharide fragments. All the structures are in global minimum except NeuNAca(2–9)NeuNAc. Grouped are the common electronegative atoms occupying similar positions: ——, NeuNAca(2-3)Gal; ............, NeuNAca(2-6)Gal; - - - - - - , NeuNAca(2–8)NeuNAc; . . . . . , NeuNAca(2–9)NeuNAc.
atoms of the second residues. The location of water is more or less equivalent in all the structures. The conserved water-mediated interactions may help to stabilize the conformation and thereby maintaining the structural similarity. It is also of interest to observe that the O2 of the galactose residue in NeuNAca(2-3)Gal and the O9 of the second NeuNAc residue in NeuNAca(2-8)NeuNAc occupying similar positions are having water-mediated hydrogen bonding interaction with O7 of the first sialic acid. Though no corresponding electronegative atoms are present in that position in NeuNAca(2-6)Gal and in NeuNAca(29)NeuNAc, examination of these structures reveals that the water at the equivalent location is mediating hydrogen bonds between O7/O8 of the first sugar and O5/O4 of the second sugar in NeuNAca(2-6)Gal. And in NeuNAca(2-9)NeuNAc, similar type of water-mediated interaction is observed between O7 of first sialic acid and O8 of the second sialic acid. In all these structures, the water that mediate the hydrogen bonds between the residues may have a role in maintaining the structural similarity, and this maintain a complementary shape for neuraminidase activity. However, it is hard to obtain dynamical nature of the watermediated hydrogen bonding in hard sphere energy calculations. It is also noticed that in the modeled neuraminidase– sialyloligosaccharide complexes, the side chain of the conserved Asp151 occupies the position of the water which mediated the hydrogen bonding interaction
between the residues of sialyloligosaccharides. This is a clear indication that water maintains the conformation of the linkages in the free form, which is crucial for neuraminidase cleavage. On entering into the active site, the water will be replaced by the side chain of the conserved Asp151, facilitating the enzyme to cleave the terminal sialic acid.
4. Conclusions The molecular dynamics studies reveals that the terminal disaccharide fragments of sialyloligosaccharides shares the common conformational space for the glycosidic torsions even though the linkages are different. Among the regions allowed, the global minimum lies in the conformational region Bi. The local minima occur in the Ai region for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, and NeuNAca(2-8)NeuNAc and Ai and Ci regions for NeuNAca(2-9)NeuNAc. The conformational preference of sialyloligosaccharides reveals a similarity between these structures, which an essential requirement for neuraminidase specificity towards sialyloligosaccharides of distinct linkages. The local change in the conformation of the active site residues may contribute for the specificity difference between linkages, however, the flexibility in the conformations can also have role. However, future docking studies of these linkages with specific receptors of distinct origin will help to substantiate this study.
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Acknowledgements The National Facility for High Resolution Graphics housed at Bioinformatics Centre, Madurai Kamaraj University is gratefully acknowledged. Support from the Department of Science and Technology (DST Grant NO. SP/SO/D-14/99) is acknowledged.
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Veluraja, K., Rao, V.S.R., 1980. Theoretical studies on the conformation of b-D-N-acetyl neuraminic acid (sialic acid). Biochim. Biophys. Acta. 630, 442–446. Veluraja, K., Rao, V.S.R., 1983. Theoretical studies on the conformation of monosialogangliosides and disialogangliosides. Carbohydr. Polymers 3, 175–192. Veluraja, K., Rao, V.S.R., 1984. Studies on the conformations of sialyloligosaccharides and implications on the binding specificity of neuraminidases. J. Biosci. 6, 625–634. Veluraja, K., Suresh, M.X., Christlet, T.H.T., Rafi, Z.A., 2001. Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase. J. Biomol. Struct. Dyn. 19, 33–45. von Itzstein, M., Colman, P., 1996. Design and synthesis of carbohydrate-based inhibitors of protein–carbohydrate interactions. Curr. Opin. Struct. Biol. 6, 703–709. Weiner, S.J., Kollman, P.A., Case, D.A., Singh, U.C., Ghio, C., Alagona, G., Profeta Jr., S., Weiner, P., 1984. A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc. 106, 765–784. Weiner, S.J., Kollman, P.A., Nguyen, D.T., Case, D.A., 1986. An all atom force field for simulation of proteins and nucleic acids. J. Comput. Chem. 7, 230–252. Woods, R.J., 1998. In: Schleyer, P.V.R. (Ed.), Encyclopedia of Computational Chemistry. Vol 1. Wiley, Chichester, pp. 220–233. Yamasaki, R., Bacon, B., 1991. Three-dimensional structural analysis of the group B polysaccharide of Neisseria meningitidis 6275 by two-dimensional NMR: The polysaccharide is suggested to exist in helical conformations in solution. Biochemistry 30, 851–857.
Conformations of terminal sialyloligosaccharide fragments — a molecular dynamics study M. Xavier Suresh, K. Veluraja* Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627 012, India Received 30 May 2002; received in revised form 14 November 2002; accepted 31 December 2002
Abstract Molecular dynamics simulations have been performed to understand the conformational features of the terminal sialyloligosaccharide fragments NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc. The conformational regions Ai ; Bi and Ci were identified in the Ramachandran plot. Analysis of the 1000 ps trajectories collected through simulation (2000 ps in the case of NeuNAca (2-9)NeuNAc) revealed that these molecules have conformational propensity in region Bi. The occurrence of these molecules in the common conformational space leads to a structural similarity between them. This structural similarity may be an essential requirement for the neuraminidase activity towards sialyloligosaccharides. The local change in the conformation of the active site residues of neuraminidases may contribute for the specificity differences between different linkages of sialyloligosaccharides. A highly conserved water-mediated hydrogen bond observed in these structures between the sugar residues, acts as an additional stabilizing force. r 2003 Published by Elsevier Science Ltd. Keywords: Sialyloligosaccharide; Molecular dynamics; Structural similarity
1. Introduction Cell surface carbohydrates deriving from glycolipids, glycoproteins and proteoglycans mediate the tight associative interactions between molecules and cells or between cells and cells at the origin of numerous recognition processes (Varki, 1993; Dwek, 1996). The role of carbohydrates in biological recognition is known to be of great importance, not now but for quite a long time. The first indication of such a role came from the discovery by Hirst (1942a, b) that the influenza virus agglutinates erythrocytes. It was later shown by Gottschalk (1957) that this virus binds to the red cells through sialic acid residues present on the cell surface. Cell surface carbohydrates are responsible for such phenomena as infectious interaction, embryonic development, blood group specificity and clearance of senescent cells (Hughes, 1976; Jeanloz and Codington, 1976; Sharon, 1979; Holmgren et al., 1980).
*Corresponding author. Tel.: +1-91-462-333887; fax: +1-91-462322973. E-mail address: [email protected] (K. Veluraja). 0022-5193/03/$ - see front matter r 2003 Published by Elsevier Science Ltd. doi:10.1016/S0022-5193(03)00050-X
Sialic acid is an unusual acidic amino sugar present in glycolipids, glycoproteins, oligosaccharides and polysaccharides. It is one of the important building blocks of gangliosides and play a vital role in a variety of fundamental physiologically and pathologically important processes (Rosenberg, 1995; von Itzstein and Colman, 1996; Reutter et al., 1997; Mammen et al., 1998). Numerous derivatives of sialic acids have been identified (Kelm and Schauer, 1997; Schauer et al., 1997), of which N-acetylneuraminic acid (NeuNAc) is the most abundant one. Very small quantities of sialic acids are also found in a free state. Though sialic acid occurs as the a-anomer in gangliosides, the b-anomer is predominant in solution; the b-anomer has also been found in the crystalline state (Flippen, 1973; Bhattacharjee et al., 1975). They occur terminally or in buried form in sialyloligosaccharides. An analysis across the CCSD, a database for complex carbohydrate structures, shows that the sialic acid predominantly occurs as a terminal sugar in the complex carbohydrate structures (Doubet et al., 1989; Christlet et al., 2000). The terminal sialic acid is attached to the penultimate sugar with specific type of linkages. The predominant sialic acid linkages that are prevalent in sialyloligosaccharides are:
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NeuNAca(2-3)Gal–R; NeuNAca(2-3)GalNAc–R; NeuNAca(2-6)Gal–R; NeuNAca(2-6)GalNAc–R; NeuNAca(2-8)NeuNAc–R and NeuNAca(2-9)NeuNAc–R (Varki et al., 1999), where the R indicates the penultimate bulky chains of the specific type of glycosidic linkage. Studies indicate that the sialyloligosaccharide fragments are the substrates for many enzymes such as neuraminidases (Corfield et al., 1981, 1983; Suzuki et al., 1986; Baker et al., 1987; Daniels et al., 1987; Kopitz et al., 1996; Gornati et al., 1997) that cleave terminal sialic acids. Influenza virus neuraminidase can accommodate a minimum of two residues alpha ketosidically linked with sialic acid (Veluraja et al., 2001). And Endoneuraminidase N, derived from the Escherichia coli K1-specific bacteriophage PK1, requires a minimum of eight a2,8-linked sialic acids for binding and cleaves poly sialic acid into oliogomers of two to seven residues (Finne and Makela, 1985; Hallenbeck et al., 1987). They are the receptor site for many biological macromolecules and are involved in various life processes. The extreme structural diversity possible for oligosaccharides is the key to their postulated biological roles. Carbohydrate structural and conformational analysis is a prerequisite for further studies involving carbohydrate–protein interactions which are important, e.g., in protein trafficking, inflammation or bacterial infection processes. The analysis may be divided into the analysis of the primary structure as well as the three-dimensional structure. The former deals with the identity of the constituent monosaccharides, substituents if any, and the sequence of the monosaccharide residues and the latter includes conformation, flexibility and dynamics which are important properties to investigate in order to understand the function of carbohydrates in relation to other molecules, most often proteins. It is obvious that a detailed knowledge of the structure and dynamics of carbohydrates, both free and bound to proteins is indeed relevant (Bush et al., 1999). In order to gain information about the probable conformation and dynamics of the oligosaccharides, the molecular dynamics technique has been used. It can also assist in the interpretation of NMR data, in the study of conformational flexibilities, in the analysis of the conformational variability of the glycosidic linkages, etc., other than the efficient searches of low-energy structures in the case of complex carbohydrates. As sialic acid is the sugar found at the non-reducing end of glycolipids and glycoproteins, and is a cleavage product of the sialyloligosaccharides by the sialidases or neuraminidases, investigation of the conformational dynamics of these sialyloligosaccharide fragments is essential to understand its molecular recognition. The conformation of oligosaccharides and polysaccharides containing the disaccharides moieties NeuNAca(23)Gal, NeuNAca(2-6)Gal or NeuNAca(2-8)NeuNAc
as their part have been studied in the past by various other groups using NMR spectroscopy and computational methods (Breg et al., 1989; Blanco et al., 2000; Acquotti et al., 1990; Poppe et al., 1989; Baumann et al., 1993; Yamasaki and Bacon, 1991; Veluraja and Rao, 1983; Mukhopadhyay and Bush, 1994; Vasudevan and Balaji, 2002). The present work aims at having a complete picture about the conformational preferences and flexibility of the four disaccharide fragments, NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(28)NeuNAc and NeuNAca(2-9)NeuNAc that are prevalently observed in sialyloligosaccharides using molecular dynamics simulations over a period of 1000 ps with the explicit inclusion of water molecules. 2. Materials and methods 2.1. Initial model In the present study, the disaccharide fragments of sialyloligosaccharides that have been considered are NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(28)NeuNAc and NeuNAca(2-9)NeuNAc and their structures are depicted in Fig. 1. The internal parameters such as bond length, bond angle and torsion angle which were obtained from the standard geometry in 2C5 chair form (Flippen, 1973; Veluraja and Rao, 1980) were used for the generation of the coordinates for the sialic acid and hence the sialyloligosaccharide fragments. 2.2. Construction of glycosidic linkages to form sialyloligosaccharide fragments While the construction of glycosidic linkages to form sialyloligosaccharide fragments, the net effect was the removal of a molecule of water upon forming the glycosidic linkage. It is necessary to take care of the allowed conformational flexibility of the disaccharide fragments, which arises due to the freedom of rotation around glycosidic torsions and the exocyclic bond. The definitions for these dihedral angles (Fig. 1) are as follows: (a) NeuNAca(2-3)Gal: fg ¼ C1 C2 O2 C3; cg ¼ C2 O2 C3 H3: (b) NeuNAca(2-6)Gal: fg ¼ C1 C2 O2 C6; cg ¼ C2 O2 C6 H61; w ¼ O2 C6 C5 O5 (H61 is that hydrogen which makes an angle of H61–C6–C5–O5=120 when w=0 )
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Fig. 1. The structure of terminal sialyloligosaccharides together with the torsions labeled: (a) NeuNAca(2-3)Gal, (b) NeuNAca(2-6)Gal, (c) NeuNAca(2-8)NeuNAc, and (d) NeuNAca(2-9)NeuNAc.
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(c) NeuNAca(2-8)NeuNAc: fg ¼ C1 C2 O2 C8; cg ¼ C2 O2 C8 H8; w2 ¼ C9 C8 C7 C6; w1 ¼ C8 C7 C6 C5 (d) NeuNAca(2-9)NeuNAc : fg ¼ C1 C2 O2 C9; cg ¼ C2 O2 C9 H91; w3 ¼ O2 C9 C8 C7; w2 ¼ C9 C8 C7 C6; w1 ¼ C8 C7 C6 C5 (H91 is that hydrogen which makes an angle of H91-C9-C8-C7=120 when w3 =0 ). The glycosidic angle is fixed at 117.5. The carboxylic acid group of sialic acid is considered to be ionized in neutral pH and hence the total charge per sialic acid is 1. The charge assigned to the atoms around the carboxylic acid group of sialic acid are C2=0.379 and C1=0.679. The charge associated with each of the oxygen atoms of C1 is 0.792. No counter ion is included. All the hydrogen atoms were treated explicitly and all-atom simulations have been performed.
The water molecules were added from the library of the modeling software Insight II. The numbers of water molecules differ from structure to structure. The resulting ensemble was energy minimized using steepest descent and conjugate gradient methods until the ( 1. The potential gradient reduces to 0.01 kcal mol1A temperature of the system is maintained at 300 K. The width of the integration steps of the MD simulations was 1fs at a 64-bit precision of the computer word. Non-bonded interactions were calculated on the basis of ( under periodic atom pair with a cut-off radius 20 A boundary conditions. The history of information was recorded for every 500 steps of trajectory. The whole system is subjected to MD simulations for a period of 1000 ps were performed followed by a 30 ps equilibration except for NeuNAca(2-9)NeuNAc. The simulation period is extended to 2000 ps for NeuNAca(2-9)NeuNAc considering the flexibilities of the exocyclic torsions. To examine the structural and conformational variations during these simulations, structures from the respective local minima were selected systematically. And the selected structures were further energy minimized using steepest descent method for 100 cycles with a subsequent conjugate gradient method until the energy ( 1. The MD gradient dropped below 0.01 kcal mol1 A trajectory informations were analyzed using ANALYSIS module of Biosym package.
2.3. Computational procedures 3. Results and discussion The molecular modeling studies were carried out on a Silicon Graphics IRIS Crimson Elan graphics workstation using Biosym, a model building package by Molecular Simulations Inc. (MSI). Insight II was the graphical interface. Molecular mechanics calculations were carried out in the AMBER force field which incorporates carbohydrate force field (Weiner et al., 1984; Weiner et al., 1986; Homans, 1990) using DISCOVER module (an energy minimization program) by MSI. The distance-dependent dielectric constant e ¼ 4rij was used. When simulating a microscopic system of finite size, the boundary of the system should be treated such as to minimize edge effects the standard procedure is to use periodic boundary conditions. The biomolecule and its surrounding solvent molecules are put into a periodic space-filling box, which is treated as if it is surrounded by its identical translated images of itself. In ( is this present study, a box of dimension 20 20 20 A constructed for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal and NeuNAca(2-8)NeuNAc and the solvated sialyloligosaccharide fragments are placed at the center. For the disaccharide NeuNAca(2-9)NeuNAc which is likely to have larger extension, hence the box size of ( was considered. This can facilitate the 25 25 25 A free movement of the molecule during the dynamics.
In our attempt for the detailed conformational analysis of the solvated carbohydrates, especially of terminal disaccharide fragments of sialyloligosaccharides, we performed a series of different extended MD simulations. The incorporation of water in the simulations is mandatory for a reasonable description of conformations and entropy. Raising the temperature distorts the ring geometry of sialic acid, and hence the simulations with temperature 300 K were considered for further analysis. 3.1. Disaccharide NeuNAca(2-3)Gal Molecular dynamics was carried out for the disaccharide NeuNAca(2-3)Gal with explicit water over a period of 1000 ps with a history of structures at every 0.5 ps which leads to a total of 2000 structures. Fig. 2 shows the distribution of glycosidic torsions in the (fg ; cg ) space for the disaccharide, from this simulation. From the figure it is clear that there are two distinct regions in the conformational space indicated as region A1 and region B1. A total of 100 structures selected from these regions were energy minimized, only the relative energies of global and local minimum conformations are
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Fig. 2. The (fg ; cg ) distribution in NeuNAca(2-3)Gal. The allowed regions are noted as A1 and B1. Table 1 Minimum energy conformers for NeuNAca(2–3)Gal No.
f (deg)
c (deg)
Relative energy (kcal mol1)
1 2
66 164
10 59
0.0 3.0
given in Table 1. It is clear from the table that region B1 is the global minimum energy region and region A1 is a local minimum energy region and the energy difference being 3 kcal mol1. In the earlier work using the hard sphere energy calculations, Veluraja and Rao (1983, 1984) suggested that there are three conformational regions possible for this NeuNAca(2-3)Gal linkage. In another work, Mukhopadhyay and Bush (1994) reported four local minimum regions. However, the global minimum conformations observed in these studies are also around the region that is same as that of the region B1 as in the present study and the other high-energy regions are not observed in this present study. It is also to be mentioned here that the terminal NeuNAca(2-3)Gal in ganglioside GD1a also favor the conformational region B1 (Veluraja and Rao, 1983). Earlier work on the conformational aspects of GM4 indicates that there are three allowed conformational regions accessible for the same fragment NeuNAca(2-3)Gal with the minimum in region A1 (Poppe et al., 1989). In the same study, the NOE experimental data showed that the population is more (about 60%) in region B1. For the NeuNAca(2-3)Gal fragment in GM1, Acquotti et al. (1990) were able to get a conformation in region A1 using NOE distance– mapping procedure. Similar conformational preference is also observed in the earlier hard sphere energy
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calculations (Veluraja and Rao, 1983). However, this conformational preference has already been accounted for the vicinal location of the GalNAc residue attached to the galactose residue. Hence, the fragment in GM1 cannot be equated to the terminal fragments. In the present molecular dynamics study, of the two conformational regions accessible, the region B1 is more populated as evidenced from the dynamics trajectory which is in accordance with the NOE data of Poppe et al. (1989). Even though various starting conformations for the molecule from the other regions given, the molecule tend to be in the region B1 and the transitions are observed only between the A1 and B1 more frequently. A representation plot from the dynamics trajectory clearly shows the transitions between regions A1 and B1 (Fig. 3a). The starting conformation for this plot is from region B1. The dynamics has been further extended over a period of 3000 ps and no such transitions to the regions other than A1 and B1 have been observed (data not shown). The absence of accessibility of the conformational space in the third region (as identified by hard sphere energy calculations) other than A1 and B1 may be due to the high-energy barrier separating these regions. The global minimum energy conformation of NeuNAca(2-3)Gal is well stabilized by water-mediated hydrogen bonds (Fig. 4). The significance of the watermediated hydrogen bonding interactions in the global minimum conformational space has been brought into light by an exhaustive analysis over the complete 1000 ps dynamics trajectory. It reveals that in more than 50% of the structures, a molecule of water is invariably present at a particular position and do mediate a hydrogen bond interaction between O7 of NeuNAc and O2 of Gal, whenever this disaccharide assumes the conformations in the global minimum energy region B1. Even though different water occupies that particular position, the position is highly conserved. 3.2. Disaccharide NeuNAca(2-6)Gal Analysis of the MD trajectory records of the simulated structures of NeuNAca(2-6)Gal reveals interesting features. Fig. 5 depicts the dynamics behavior of the glycosidic torsions of NeuNAca(2-6)Gal in solvent environment over a period of 1000 ps molecular dynamics run. From the figure it is inferred that there are two conformational regions available, region A2 and B2. The regions are roughly similar to the one that has been found for the disaccharide NeuNAca(2-3)Gal. The relative energies for the different conformational regions were worked out by systematically sampling the structures from the trajectory records as mentioned earlier (100 structures). The possible conformations and their relative energies are given in Table 2. If we have
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Fig. 3. (a-d) The representative plots of MD trajectory showing the transitions of the glycosidic torsions fi ; ci for the disaccharide fragments.
a closer look into Table 2, it is noticed that in the minimum energy conformation the glycosidic torsion (fg ; cg ) favor values around (52 , 88 ) which is in the region B2. An energy difference of 13 kcal mol1 is noticed between region A2 and region B2. And hence it is clear from the table that the conformational propensity for these molecules is in region B2.
It is interesting to observe, in this disaccharide NeuNAca(2-6)Gal, the behavior of the exocyclic torsion (w), since it provides an additional freedom of rotation and hence enhances the flexibility of the molecule. From Table 2 it is found that the exocyclic torsion w for the minimum energy conformation is 73 . But it can also assume other possible staggered values (60 and 180 )
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395
Fig. 4. The global minimum energy conformation of NeuNAca(2-3)Gal with the typical water-mediated interaction.
population with different w values, w ¼ 60 ; 180 , 60 ; are 83%, 4%, 13%, respectively, gives a clear indication of the predominance in +60 conformation. Analysis of the structures in the global minimum energy conformational space indicates that a watermediated hydrogen bonding interaction is formed either between O7 of the first residue and O5 of the second residue (Gal) or between O8 of the first residue and O4 of the second residue. However, the former is more prevalent than the later. A representative structure in the global minimum energy conformation with watermediated hydrogen bonding interaction is shown in Fig. 6. This water approximately occupies the same location in space as in the case of NeuNAca(2-3)Gal. 3.3. Disaccharide NeuNAca(2-8)NeuNAc
Fig. 5. The (fg ; cg ) distribution in NeuNAca(2-6)Gal. The allowed regions are noted as A2 and B2.
Table 2 Minimum energy conformers for NeuNAca(2–6)Gal No.
f (deg)
c (deg)
w (deg)
Relative energy (kcal mol1)
1 2 3 4 5
52 60 178 69 179
88 74 55 36 51
73 57 170 172 68
0 8.5 13.4 14.3 22.3
with possibility of an increase in energy (Table 2). For example, a difference of 8.5 kcal mol1 is accounted when a switching occurs from values near +73 to values around 57. It is important to note that in the earlier hard sphere energy calculations the favored value for w is also around 73 . An analysis of the available
The molecular dynamics calculations that reveal the glycosidic conformational flexibility of NeuNAca(28)NeuNAc is shown in Fig. 7. From the figure it is clear that the molecule can be in two regions, region A3 and region B3. The conformation of the molecule occurred for longer duration is in region B3. Minimization of the selected structures reveals that the global minimum is in the region B3 (Table 3). The glycosidic torsion along with the exocyclic rotations involved in this linkage (fg ; cg ; w2 ; w1 ) prefers values around (63 , 5 , 60 , 169 ) in the global minimum energy conformation. These are in agreement with the conformations deduced earlier based on energy calculations (Veluraja and Rao, 1984). A difference of 6 kcal mol1 is noticed between the region A3 (local minimum) and region B3 (global minimum). Recently, Vasudevan and Balaji (2002) explored the conformational details of the disialosyl fragment NeuNAca(2-8)NeuNAc using molecular dynamics for a period of 1 ns with another force field, GROMOS96 (Gunsteren and Berendson, 1996). As evidenced from
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396
Fig. 6. The global minimum energy conformation of NeuNAca(2-6)Gal with the typical water-mediated interaction.
hydrogen bonding interactions. Carefully analyzing the water-mediated interactions, it is interesting to note that there is a water molecule that has been present at a hydrogen bonding distance from O7 of the first residue is being interacting with O9 of the second residue. That is, it has been involved in the water-mediated hydrogen bonding interaction between the two sugar residues and thereby stabilizing the structure of the disaccharide. The global minimum energy conformation with watermediated hydrogen bonding interaction is depicted in Fig. 8. 3.4. Molecular dynamics of disaccharide NeuNAca (2-9)NeuNAc
Fig. 7. The (fg ; cg ) distribution in NeuNAca(2-8)NeuNAc. The allowed regions are noted as A3 and B3.
Table 3 Minimum energy conformers for NeuNAca(2-8)NeuNAc No. f (deg) c (deg) w2 (deg) w1 (deg) Relative energy (kcal mol1) 1 2 3 4
63 163 47 56
5 17 16 21
60 93 58 92
169 176 65 176
0.0 5.8 7.8 9.1
earlier studies that the use of the different force fields do not significantly affect the overall conformation of the oligosaccharides despite little variations (Qasba et al., 1994; Woods, 1998; Vasudevan and Balaji, 2002). The structures in minimum energy conformations are stabilized largely by inter residue water-mediated
The 2000 ps molecular dynamics simulation of NeuNAca(2-9)NeuNAc reveals that the glycosidic conformational angle of this disaccharide fragment prefers three distinct conformational space in the (fg ; cg ) Ramachandran plot which are designated as regions A4, B4 and C4 (Fig. 9). The energy minimization results of the 200 systematically selected structures (collected for every 10 ps) are shown in Table 4. It is obvious from the table that in the global minimum, the glycosidic conformation occurs in the allowed region B4. However, when the conformation is shifted to the allowed regions A4 and C4, the energy of the molecule increases to 5.8 kcal mol1 (conformer 3) and 12.0 kcal mol1 (conformer 8). The high-energy region C4 is accessible only for a very little period in the dynamics trajectory. The conformational preference of the exocyclic torsional angle involved in the linkage reveals that w1 tends to favor a trans conformation (w1 E171 ). In the minimum energy conformations, w2 and w3 favor values around 180 , 760 . An inspection of the time plots of w2 and w3 (Fig. 10a and b) reveals that all the three staggered conformations are accessible for these two torsions however, in the major period of simulated time w2 favors
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Fig. 8. The global minimum energy conformation of NeuNAca(2-8) NeuNAc with the typical water-mediated interaction.
Fig. 9. The (fg ; cg ) distribution in NeuNAca(2-9)NeuNAc. The allowed regions are noted as A4, B4 and C4.
a value of 60 and w3 favors a value of +60 and 180 . w2 can also favor a value of +60 and w3 ; 60 with a lesser period of time in the simulation which is also reflected in the minimum energy conformations. When w2 and w3 prefer values around 60 , the relative energy of the system is 5.8 kcal mol1 and when w2 and w3 prefer values around +60 , the relative energy is 5.9 kcal mol1. However, when w1, w2 and w3 takes up a trans conformation (wi’s =180 ), the conformational energy increases to 26.1 kcal mol1 (conformer 10).
In the global minimum energy conformation direct and water-mediated hydrogen bonding plays a predominant role in the stabilization of the structure (Fig. 11). In this conformation a direct hydrogen bonding interaction is observed between O9 of the first sialic acid and the acetamido carbonyl of the second sialic acid and between O8 of the first sialic acid and carboxyl of the carboxylic acid group of the second sialic acid. A water also bridges the disialic acid by mediating a hydrogen bond between O7 of first sialic acid and the carbonyl of the carboxylic acid of second sialic acid. When the torsional angle w3 in the global minimum conformation is shifted from 173 to 98 (conformer 2, energy difference 4.5 kcal mol1), the hydrogen bonding pattern changes completely. In this conformation only a single hydrogen bond is possible. The hydrogen bond can be either a direct, between O7 of the first sialic acid and the N5 of the second sialic acid or a water mediated hydrogen bond between these two atoms. The loss of two hydrogen bonds perhaps accounts for this energy difference. The structures in other conformations have also been stabilized by hydrogen bonding interactions. An earlier investigation based on molecular modeling of this substrate into the active site of influenza virus N9 neuraminidase (Veluraja et al., 2001) revealed that the conformational parameter that the enzyme can accommodate at its binding pocket are (fg ; cg ; w1 ; w2 ; w3 )= (73, 49, 165, 176, 162). A conformational structure close to the above parameter is observed in our molecular dynamics calculation (conformer 10), but the energy of the conformer is about 26.1 kcal mol1 higher than that of the global minimum. However, this conformer differs from the global minimum energy
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Table 4 Minimum energy conformers for NeuNAca(2-9)NeuNAc No.
f (deg)
c (deg)
w3 (deg)
w2 (deg)
w1 (deg)
Relative Energy (kcal mol1)
1 2 3 4 5 6 7 8 9 10
67 45 175 78 173 68 63 64 62 58
68 76 47 62 45 44 65 72 64 66
173 98 61 49 168 50 60 54 59 175
62 57 53 53 56 61 176 92 175 170
174 179 170 157 174 165 179 163 176 176
0.0 4.5 5.8 5.9 6.8 7.7 7.8 12.0 23.3 26.1
Fig. 10. The time plot showing (a) the preference of w2 and (b) the preference of w3 :
conformer only in the favored value of w2 : Hence, there is a possibility that the enzymes may take the global minimum energy structure in the binding pocket and subsequently shift the conformation of w2 value from 170 to 60 before cleaving it.
3.5. Comparison of the conformations of disaccharide fragments An in-depth analysis of the conformational dynamics of the disaccharides reveals that the distribution of the
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Fig. 11. The global minimum energy conformation of NeuNAca(2-9)NeuNAc showing the direct and water-mediated hydrogen bonding interactions.
glycosidic conformations (fg ; cg ) of the disaccharide fragments of sialyloligosaccharide NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc, are roughly similar. It is clear from the molecular dynamics of the (fg ; cg ) plots, the disaccharide fragments of sialyloligosaccharides shares a common conformational space for glycosidic torsion irrespective of the type of linkages. The distributions have been restricted mainly into two allowed regions Ai and Bi (i=1–4) in the Ramachandran plane and an additional region C4 for NeuNAca(2-9)NeuNAc, which is a high-energy region (12.0 kcal mol1) and is accessible for a relatively smaller duration. For these disaccharides the global minimum has been observed in the region Bi. The local minima occur in region Ai, but the energy difference between the two regions for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca (2-8)NeuNAc and NeuNAca(2-9)NeuNAc is about 3.0, 13.4, 5.8 kcal mol1, and 5.8 kcal mol1, respectively. However, these molecules needs to cross barriers of 15, 33, 20 and 20 kcal mol1, respectively, to have transition from region Bi to region Ai. The transitions between these regions are found to be non-periodical and a representative plots of MD trajectory for the glycosidic torsions fi ; ci for the disaccharide fragments are shown in Figs. 3a–d. When computation was carried out using the other force field parameter (CVFF consistent valence force field) there is a shift in the magnitude of relative energy difference to the order of +1.5 kcal mol1 between the global minimum and local minima when compared with AMBER force field parameters and may not influence the conformational preference.
Further analysis on the favored conformations of these disaccharide fragments as a function of glycosidic torsion and exocyclic bond reveals a structural similarity and is schematically shown in Fig. 12. The conformers corresponds to this structural similarity are in global minimum except for of NeuNAca(2-9)NeuNAc (conformer 10). Water-mediated interactions that stabilize the structures also maintains the structural similarity. This type of structural similarity due to conformational dynamics plays a significant role in neuraminidase specificity as proposed earlier (Veluraja and Rao, 1984; Suresh and Veluraja, 2000; Veluraja et al., 2001). The finding by Kobasa et al. (1999) suggests that the adaptation of neuraminidase to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of influenza virus A neuraminidase in and around the active site to facilitate the binding of different species of sialic acid. For example, neuraminidase of N2 human influenza viruses isoleucine at position 275 is associated with high activity for NeuNAca(2-3)Gal and very low activity for NeuNAca(2-6)Gal. A change to valine results in higher NeuNAca(2-6)Gal activity without affecting NeuNAca(2-3)Gal activity. Even though the change in the conformations of the active site residues greatly alter the specificity difference of various substrates, the structural similarity in the conformations of the sialyloligosaccharides likely to play a vital role in cleavage. When these disaccharide structures favors the structural similarity, the water at a certain location has been observed to have an interaction with O7 of the first sialic acid as well as with the appropriate electronegative
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Fig. 12. The schematic representation of structural similarity observed among the disaccharide fragments. All the structures are in global minimum except NeuNAca(2–9)NeuNAc. Grouped are the common electronegative atoms occupying similar positions: ——, NeuNAca(2-3)Gal; ............, NeuNAca(2-6)Gal; - - - - - - , NeuNAca(2–8)NeuNAc; . . . . . , NeuNAca(2–9)NeuNAc.
atoms of the second residues. The location of water is more or less equivalent in all the structures. The conserved water-mediated interactions may help to stabilize the conformation and thereby maintaining the structural similarity. It is also of interest to observe that the O2 of the galactose residue in NeuNAca(2-3)Gal and the O9 of the second NeuNAc residue in NeuNAca(2-8)NeuNAc occupying similar positions are having water-mediated hydrogen bonding interaction with O7 of the first sialic acid. Though no corresponding electronegative atoms are present in that position in NeuNAca(2-6)Gal and in NeuNAca(29)NeuNAc, examination of these structures reveals that the water at the equivalent location is mediating hydrogen bonds between O7/O8 of the first sugar and O5/O4 of the second sugar in NeuNAca(2-6)Gal. And in NeuNAca(2-9)NeuNAc, similar type of water-mediated interaction is observed between O7 of first sialic acid and O8 of the second sialic acid. In all these structures, the water that mediate the hydrogen bonds between the residues may have a role in maintaining the structural similarity, and this maintain a complementary shape for neuraminidase activity. However, it is hard to obtain dynamical nature of the watermediated hydrogen bonding in hard sphere energy calculations. It is also noticed that in the modeled neuraminidase– sialyloligosaccharide complexes, the side chain of the conserved Asp151 occupies the position of the water which mediated the hydrogen bonding interaction
between the residues of sialyloligosaccharides. This is a clear indication that water maintains the conformation of the linkages in the free form, which is crucial for neuraminidase cleavage. On entering into the active site, the water will be replaced by the side chain of the conserved Asp151, facilitating the enzyme to cleave the terminal sialic acid.
4. Conclusions The molecular dynamics studies reveals that the terminal disaccharide fragments of sialyloligosaccharides shares the common conformational space for the glycosidic torsions even though the linkages are different. Among the regions allowed, the global minimum lies in the conformational region Bi. The local minima occur in the Ai region for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, and NeuNAca(2-8)NeuNAc and Ai and Ci regions for NeuNAca(2-9)NeuNAc. The conformational preference of sialyloligosaccharides reveals a similarity between these structures, which an essential requirement for neuraminidase specificity towards sialyloligosaccharides of distinct linkages. The local change in the conformation of the active site residues may contribute for the specificity difference between linkages, however, the flexibility in the conformations can also have role. However, future docking studies of these linkages with specific receptors of distinct origin will help to substantiate this study.
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Acknowledgements The National Facility for High Resolution Graphics housed at Bioinformatics Centre, Madurai Kamaraj University is gratefully acknowledged. Support from the Department of Science and Technology (DST Grant NO. SP/SO/D-14/99) is acknowledged.
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