Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase.

Transcription

1 Chapter 3 Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase. 3.1 Introduction Neuraminidases or sialidases, (EC , acylneuraminyl hydrolase) catalyze the removal of terminally occurred a-ketosidically linked sialic acid (SA) from sialyloligosaccharides (SOS) of various glycoconjugates and play roles in pathogenesis, bacterial nutrition and cellular interactions (Kopitz et al., 1996; Gornati etal., 1997; Corfield etal., 1981; 1983; Daniels etal., 1987; Suzuki et al., 1986). In addition to their role in the removal of SA from glycoproteins and glycolipids, mammalian neuraminidases have been shown to modulate the SA profile of cells, thus regulating the immune system (Pilatte et al., 1993) and controlling the half lives of circulating cells (Bratosin et al., 1995), apoptosis (Peter et al., 1995; Richard et al., 2002) and are essential

2 determinants for maintaining the glycoproteins in blood, and in the binding of toxins and viruses to cells (Colman et al., 1983). The neuraminidases of viruses are thought to enhance the viral mobility via the hydrolysis of the a(2-3)- or a(2-6)- glycosidic linkages between a terminal SA and its adjacent carbohydrate moiety on the host receptor (Gottschalk, 1957; 1959). Accordingly, inhibitors of such neuraminidases should block viral and bacterial pathogenesis and should serve as drugs in infectious processes (Sabesan etal., 1995; De Clercq, 2002; Englund, 2002; Colman, 2002). The cleavage of naturally occurring SOS by neuraminidases partially depends on the nature of the penultimate sugar and the oligosaccharide sequence (Shukla and Schauer, 1986). The types of SA linkages that are commonly found in SOS are NeuNAca(2-3)Gal - R; NeuNAca(2-3)GalNAc - R; NeuNAca(2-.6)Gal - R; NeuNAca(2-6)GaINAc - R; NeuNAca(2-8)NeuNAc - R; NeuNAca(2-9)NeuNAc - R, where R indicates the penultimate bulky chains of the specific type of glycosidic linkage. As far as the specificity of linkages is concerned, most neuraminidases do not distinguish the linkages in SOS while carrying out the catalytic activity. Though neuraminidases lack linkage specificity in general, viral and bacterial neuraminidases hydrolyse the NeuNAca(2-3)Gal linkage faster than the NeuNAca(2-6)Gal linkage (Corfield et al., 1983). They also hydrolyse NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc. Influenza virus neuraminidase is one of the two glycoproteins found on the surface of viral membrane, the other being hemagglutinin (Colman and Ward, 64

3 1985). The crystal structures of neuraminidase from Influenza viruses of type A (subtype N2 & N9) and B have been reported (Colman et al., 1983; Varghese et al., 1983; Baker et al., 1987; Tulip et al., 1991; Varghese and Colman, 1991; Burmeister et al., 1992). The over all three-dimensional structures of neuraminidase determined so far are found to be similar even though they do not possess high sequence similarity (Jedrzejas etal., 1995). Three-dimensional structure of neuraminidases shows a tetrameric polypeptide for this family of proteins and each monomer has an average amino acid sequence of 388 to 392. Most of the active site and binding site residues are highly conserved (Colman et at., 1983). A few structures complexed with SA analogues have also been reported (Bossart-Whitaker et al., 1993; Burmeister et al.; 1993; Janakiraman et al., 1994; Varghese et al., 1992; von ltzstein etal., 1993; White etal., 1995; Varghese etal., 1995). The complexed structures available for influenza virus N9 neuraminidase contains the following sialic acid analogues viz., 2deoxy2,3-dehydro-N-acetyl neuraminic acid (DANA) (Bossart-Whitaker et al., 1993), a phosphonate analogue of N-acetyl neuraminic acid in a anomeric form (epana) (White et al., 1995), and 4-guanidino-2deoxy2,3-dehydro-N-acetyl neuraminic acid (GANA) (Varghese et al., 1995) at the active site. Among these complexed structures of influenza virus N9 neuraminidase, only in epana the equatorial position of SA at C2 has a bulky group and also assumes 2C 5 chair conformation which is the normal conformational preference for the SA (Veluraja and Rao, 1980) and equatorial position at C2 has a bulky group. 65

4 Though valuable information are available on the kinetics of neuraminidases and the three-dimensional structures (native as well as complexes of SA analogs) no attempt has so far been made to model SOS fragments into the active site. Usually, experimentally determined structures do not include complexes of proteins with their substrates but only of substrate analogues. Modeling substrates onto the protein is thus a vital exercise to decipher protein function and activity (Seshadri etal., 1995) and will provide extremely valuable information on the interactions between protein and substrate and specificity of neuraminidase which can further be exploited in the design of inhibitors to control the enzymatic activity of neuraminidase. In the present study, SOS fragments are modeled into the active site of neuraminidase enzyme particularly of influenza virus N9 neuraminidase to throw more light on the binding activity of neuraminidases and the conformational aspects of SOS. Molecular dynamics study carried out in this investigation serves as an ideal yardstick to estimate the stability of the modeled complex. 3.2 Materials and Methods Initial model The disaccharide fragments of SOS that have been considered for molecular modeling into the active site of the influenza virus N9 neuraminidase are NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNJAc and NeuNAca(2-9)NeuNAc. The schematic representation of these disaccharide

5 fragments together with their conformational flexibility (arises due to freedom of free rotation about the glycosidic bond and the exocyclic torsions) were shown in Figure 2.1a-d. The atom coordinates of influenza virus N9 neuraminidase used in the present study have been taken from Protein Data Bank, PDB Entry Code: 1INY (White etal., 1995). The coordinates for the SA and SOS fragments were generated in an arbitrary frame of reference using internal parameters such as bond length, bond angle and dihedral angles (Flippen, 1973; Veluraja and Rao, 1980; Arnott and Scott, 1972) Eulerian rotation The centre of the SA that is already present in the active site in its anlogue form (epana - equatorial phosphonate analogue of silaic acid - has a close analogy with SOS) was assumed as the centre of the active site. The arbitrary frame of reference was fixed by taking the centre of the SA residue of SOS as origin, C2 atom along the X-axis and C3 atom in the X-Y plane. The coordinates of SA generated in this arbitrary frame of reference has been translated into the active site of influenza virus N9 neuraminidase so that the centre of the SA residue coincides with the active site centre (Figure 3.1). Although the centers overlap, the orientation of the SA of SOS may not be equal to that of epana that is present at the active site of influenza virus N9 neuraminidase. However, a contact free allowed orientation for SA of SOS in the active site can be achieved using the principles of rigid body dynamics, by varying the Eulerian angles c1 from 0 to 360 0, from 0 to and F from 67

6 Figure 3.1 Coordinate transformation from arbitrary frame of reference to active site centre of neuraminidase

7 o to 3600 at finite increments of 100 (Figure 3.2). This leads to a total of 36x18x36 orientations for SA at the active site in the Eulerian space. By giving the Eulerjan rotations, the new coordinates obtained may be represented as a matrix representation, (x") y'j=a UY where A is the transformation matrix given by (coslvcos-cososinsjn CosWsin+cosOco$sinW sin sino A = -sinijcoscos9sincos -sinwsin4+cosocos,cos cosysine sinosincf -sin0co4 coso ) An exhaustive search have been carried out to identify the allowed Eulerian space for the SA with all possible orientations. For each orientation of the SA residue, the inter-atomic distance between the substrate (SOS fragments) and protein (influenza virus N9 neuraminidase) atoms (within 20A radius from the active site centre) were calculated. The orientation is said to be allowed if all the inter-atomic distances satisfy the Ramachandran's contact criteria (Ramachandran and Sasisekharan, 1968). The percentage of allowed orientation was calculated with respect to the total number of orientations 68

8 z Figure 3.2 Euler's angles

9 scanned in the Eulerian space, which equals Number of orientation allowed % of allowed orientation = Total number of orientations scanned Out of these allowed orientations, the best possible orientation for SA at the active site has been selected based on stereo chemical examination of the structure and favourable hydrogen bond interactions The procedure adopted to fit the disaccharide fragment of the SOS While fitting the disaccharide fragments into the active site, in addition to the Eulerian rotation, it is necessary to take care of the allowed conformational flexibility of the disaccharide fragments which arises due to the freedom of rotation around the glycosidic torsions and the exocyclic bond. The definitions for these dihedral angles are discussed in the previous chapter and are shown in Figure 2.1 The procedure adopted to fit the disaccharide fragment of the SOS is as follows: Firstly, the SA residue is kept at the best possible orientation. Then the glycosidic torsions (4g' Wg) are varied in the allowed regions of the steric maps while the bond angle of the glycosidic oxygen atom is fixed at the average value (Sundarajan and Rao, 1970). The steric maps for the disaccharide fragment of the various SOS are shown in Figure 2.2a-d and are

10 70 discussed in chapter 2. The exocyclic torsion is fixed in a staggered orientation. The contact criteria is applied so as to avoid any stereochemjcal clash with the protein atoms. The contact free region for the substrates at the active site, thus obtained by varying the above said parameters are selected as starting models for molecular mechanics calculations. The final models of molecular mechanics calculations are used for the subsequent molecular dynamics calculations Molecular mechanics calculations and dynamics 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). The N- and C- termini of the neuraminidase enzyme were kept in its ionic form. Hydrogen atoms were added to the initial PDB coordinates using standard geometry for amino acid residues taken from the Biosym package's database. Careful considerations were given while fixing the hydrogen atoms for charged amino acid residues such as lysine, arginine, glutamic acid and aspatric acid, so that their specific charges are retained. Molecular mechanics calculations were carried out in the AMBER force field which incorporates carbohydrate forcefield (Weiner et al., 1984; 1986; Homans, 1990a) using Discover module (an energy minimization program) by MSI. A distance dependent dielectric constant of was used. To reduce the computational time, a non-bonded cut off distance of 20 A was introduced. In order to eliminate any modeling artifacts, the

11 selected substrate complexed protein structure was first energy minimized using steepest descent method for 100 cycles. Subsequently, a conjugate gradient minimization was carried out until the energy gradient dropped below 0.01 kcal mo1 1 A 1 or root mean square (RMS) derivative value becomes less than or equal to 0.01 A. The energy minimized structures thus obtained were then subjected to molecular dynamics simulations over a period of 50 Ps with the coordinates recorded every 500 femto seconds. During the entire period of the simulation, the temperature was kept at 300K. The crystallographic water molecules were also included. To examine the structural variations during this simulation, MD average structure was calculated along the entire 50 Ps of production run. Finally, the resulting MD average structures were energy minimized. 3.3 Results and Discussion Fitting of Sialic acid The permissible orientations in the Eulerian space for the SA ((X-NeuNAc) at the active site of influenza virus N9 neuraminidase is shown in Figure 3.3 and in Table 3.1. It is clear from the figure that that SA predominantly restricts itself to two distinct orientational regions I ( , e , P ) and II ((D ;:e 210 0, , P ) at the active site (Table 3.1). The percentage of allowed orientations for a-neunac at the active site in the Eulerian space is found to be less than 1. 71

12 do I1_ L ' OCEP I I S 120 I -4-. I _Sig II 60 S 180 ra Figure 3.3 Allowed Eulerian space for sialic acid at the active site of influenza virus N9 neuraminidase

13 Table 3.1 Allowed spatial orientation for sialic acid at the active site Eulerian orientational D range range F range % of allowed orientation region in Eulerian space I 70-81J II * 13C * Orientation of epana as per crystallographic studies (White et al., 1995)

14 This indicates that the SA can strongly fit in a narrow region of the Eulerian space. The orientation of epana, in the Eulerian space in the complexed structure is ((t, 0, P: 130,30,100) (White et al., 1995). This orientation is roughly similar to that of the orientations in Eulerian orientational region I due to the fact that epana possesses a hydrogen at the axial position whereas a- NeuNAc possesses a carboxylic acid group at that position. When a-neunac assumes orientation similar to epana, a severe stereo chemical clash is encountered between the axial carboxylic acid group and the side chain of Tyr405. The hydroxyl oxygen of Tyr405 is at a distance of 1.7A from Cl of sialic acid. These steric hindrances caused by the carboxyl oxygens may not allow the a-neunac to occupy the orientation that is favoured by epana. The hydrogen bonding interaction between the neuraminidase and the sialic acid in orientational region I and II and the epana complex are given in Table 3.2. In both the Eulerian orientational regions the sialic acid is observed to have good number hydrogen bonding interactions with the neuraminidase. However, sialic acid in orientational region I has more hydrogen bonds compared with orientational region II Addition of second residues The first residue (SA) is fixed in the Eulerian orientational regions I and II respectively and the second residue (say Gal) is now added. For the disaccharide fragment NeuNAca(2-3)Gal, the earlier studies indicate that 72

15 Table 3.2 The important hydrogen bonding interactions of SA with influenza virus N9 neu ram in id ase Interacting SA in orientational SA in orientational epana atoms region I region II 04 0E2 G1u278: 2.6 NH1 Arg118: 3.5 0E2 G1u119: OH Tyr405: 2.6 OD2 Aspl5l: 3.5 0D2 Aspl5l: 2.9 N5 0E2 G1u278: D2 Aspi 51: 2.6 0E2 G1u278: OE1 G1u277: E2 Glu277: OE1 G1u277: NI-12 Arg293: 3.8 NH2Arg152: 3.6 ols NH2Arg371: 3.4 0E2 G1u119: 3.2 OD2Asp151: 3.3 OlD OH Tyr405: OlD NH2Arg371:

16 there exists three distinct allowed conformational regions A ( to 150 0, "41g to 0 0), B (4g to 40, 41g to 20 0) and C ( ^ g-zz 4 00 to 80 0, Wg to 0 0 ) (Veluraja and Rao, 1984). However, based on energy calculation of the isolated disaccharide fragments, it was predicted that NeuNAco(2-3)Gal disaccharide prefer region B compared to A and C (Veluraja and Rao, 1984). This is because, when it occurs in allowed region A and in allowed region C, the conformational energy increases to 4 kcal/mol and 8 kcal/mol respectively with respect to region B. Hence one can conclude that the molecule cannot occur in conformational region C because of its high energy. Even though the molecule in the conformational region A has 4 kcal/mol higher energy than that in region B, this difference may be compensated by some favorable interactions with the protein atoms when it is accommodated at the active site. Based on this, the SA of the SOS fragments fixed at various points in the Eulerian orientational regions I and II with a combination of allowed regions A and B the stereo chemistry between the protein and the substrate was checked. It is found that when the substrate goes in Eulerian orientational region I coupled with allowed regions A and B, the number of stereo chemical clashes between the substrate and the protein encountered is 0 to 3. However, when the substrate assumes Eulerian orientational region II and allowed regions A and B, the number of stereo chemical clashes encountered is more than 20. This indicates that NeuNAca(2-3)Gal can be accommodatable at the active site in Eulerian orientational region I together with conformational regions A

17 74 and B (Table 3.3). This gives rise to few structures free from stereo chemical clash, however, only one is considered for performing the molecular mechanics calculations due to the fact that all the structures are very similar as the deviation in the set of angles (t,, 'I', 0g, ij) are marginal. A similar procedure was adopted for fitting the other SOS fragments. The fitting studies indicate that NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NJeuNJAc are able to bind in the active site in Eulerian orientational region I and conformational region A without any stereo chemical clash between the substrate and protein atoms. This is the only allowed fit for these substrates. The best fit models thus obtained for the SOS fragments were considered for molecular mechanics calculations Molecular mechanics calculations for NeuNAccx(2-3)Gal and NeuNAca(2-6)Gal The molecular mechanics calculations were carried out by fixing the substrates to its best fit orientation at the active site along with the protein atoms. The energy minimized protein-.substrate complexed structure revealed that, when the substrate assumes conformation in the allowed region B, the energy of the complex is minimal. But when it takes the conformation in the allowed region A, the energy of the complexed structure increases (relative energy difference 9 kcal/mol). This clearly indicates that the enzyme can accommodate NeuNAca(2-3)Gal strictly in the conformational region B.

18 Table 3.3 Allowed orientation and conformation for NeuNAca(2-3)Gal at the active site Eulerian orientational region Conformational region Allowed! Not allowed A Allowed B Allowed [I A Not allowed 11 B Not allowed

19 Table 3.4 gives the relative energy difference between neuraminidase- NeuNAccL(2-3)Gal complex with that of the neuraminidase-neunaca(2-6)gal complex. From the table it can be inferred that neuraminidase-neunaca(2-3)gal complex is seen to have a lower energy in its bound form when compared to neuraminidase-neunacc(26)g complex by about 7 kcal/mol, indicating NeuNAca(2-3)Gal is a better binding substrate compared to that of NeuNAcct(2-6)Gal. These results concur with experiments that suggest viral neuraminidases can cleave a(2-3)-linkage faster than 2-6)-linkage (Gottschalk, 1957; 1959). Figures 3.4 and 3.5 depict stereo view of NeuNAca(2-3)Gal and NeuNAca(2-6)Gal at the active site of influenza virus N9 neuraminidase. It is interesting to note that in addition to the SA residue the hydroxyl oxygens of the second residue (Gal) are also involved in binding. They are actively taking part in several hydrogen bonding interactions with the side chains of the residues at the active site of the enzyme, implying that the second residue can act as a holding arm in the process of hydrolysis. Possible hydrogen bonds are shown in Table Molecular mechanics calculations for NeuNAca(28)NeUNA c and NeuNAc(x(2-9)NeuNAc Table 3.6 shows the results of the relative energy for both neuraminidase- NeuNAca(2-8)NeUN and neuraminidase-neu NAca(2-9) Neu NAc complex 75

20 Table 3.4 Molecular mechanics calculation of cl(2-3)- and a(2-6)-linked sialyloligosaccha ride substrates with protein atoms (The energy value is given for complexed structures) Substrate Relative energy (kcal/mol) NeuNAcct(2-3)GaI 0 NeuNAca(2-.6)Gal 7

21 x rn OC CN oty 0 4-' (I) > 0 cz 4-, 4-a ct:s C? C\J c'q rfl ' 0 Zj Cl) ) cj Z x 1 o E cz ci) =.5 c-i ci) C) >-..5; vi cz 0) C - ci) C):;:: LL

22 4r) 0 ci) 4- C,) C) > C) -c co Y-,,.o _-o 'P O ciq cfu - eo 0 (C c'.j 0 ZC) Cl) U) Z '4- o.92 > C) C C) C) 5z CO LC)> >. C") c N G) C - C) Li. o, LL 1.L

23 Table 3.5 Hydrogen bonding interaction between a(2-3)- and cx(2-6)-iinked substrates and protein atoms Substrate Protein Distance (A) Atoms Atoms NeuNAca(2-3)GaI NeuNAca(2-6)GaI 06A NH2Arg A OE2GIu A 0 H0H N5A 0E2 G1u N5A 0 H0H A 0 H0H A OD1 Asp A NEArg A NH2Arg A NH1 Arg A NH2Arg A 0 H0H SA NH1Arg OISA NH2Arg SA NH2Arg SA 0Tyr SA 0 H0H DA NH1 Arg DA NH2Arg DA NH1 Arg DA NH2Arg DA OHTyr4O A NH1Arg A OD1 Asp B NH1Arg B NH1 Arg B 0 H0H B OD1 Asp B NH2Arg B 0 H0H B NH1 Arg B 0 H0H B NEArg B NH1 Arg B NH2Arg37I B 0H0H

24 Table 3.6 Molecular mechanics calculation of a(2-8)- and cx(2-9)-iinked sialyloligosaccharide substrates with protein atoms (The energy value is given for complexes): Substrate Relative energy (kcal/mol) NeuNAca(2-9)NeuNAc NeuNAca(2-8)NeuNAc 20

25 structures. It is evident from the table that NeuNAccx(2-9)NeuNAc is a preferred substrate for binding compared to NeuNAca(2-8)NeuNAc. Figures 3.6 and 3.7 depict stereo view of NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc at the active site of influenza virus N9 neuraminidase. The hydrogen bonding interactions observed are tabulated (Table 3.7) Conformational similarity An in-depth analysis of influenza virus N9 neuraminidase-sos complexes revealed that the glycosidic conformations (g, 'yg) are roughly similar in all the substrates irrespective of the type of linkages. The conformational angles of the SOS substrates are shown in Table 3.8. Further analysis on the favored conformations of various SOS fragments including the exocyclic bond in addition to the glycosidic torsion, revealed that there exists a conformational preference, which paves a way to structural similarity. A comparison of the bound conformations of the SOS fragments NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc together with the geometry of the second residues shows the following dihedral angles: (j)g ''1kg' 02-C3-C4-05, C3-C4-05-C6 and C4-05-C6-06 (approximately -71 0, , 177 0, -59 0) in NeuNAca(2-3)Gal,, 'i' 9, 02-C6-05-C4, C6-05-C4-C3 and C5-C4-C3-H3 (approximately -88, -54, , , -63 0) in NeuNAca(2-6)Gal, 76

26 1. 0 a) U) a) > 0 cz a) -c -a (t5 0 z a) z c'.j o (I) ZD ci) z cz o a) ci) C) 0 a) CO a) -

27 c'l C) 4-, C)) ci) > 4-0 ci) -c 4-, nf! z :3 ci) z C) C\J C-) z CZ ci) ze cz o 0 Ci) C) z ci) :3 ci). 4-. > cri vi c L '4- D) '4- u_ 0 00

28 Table 3.7 Hydrogen bonding interaction between a(2-8)- and a(2-9)-iinked substrates with the protein atoms: Substrate Protein Distance (A) Atoms 04A 04A 04A N5A N5A O1OA 01 OA O1OA O1OA 07A 08A 08A 08A O1SA O1SA OISA O1SA 01 DA 01 DA 01 DA O1DA 01 DA 02A 02A O11B 09B 08B 08B 08B 07B 07B 07B N5B Atoms 0E2 G1u119 0E2 G1u278 o H0H533 0E2 G1u278 o H0H533 NE Arg152 NH1 Arg152 NH2 Arg152 o H0H533 NH2 Arg152 NH1 Arg293 NH2 Arg293 O H0H531 NH1 Arg293 NH2 Arg371 OH Tyr405 O H0H531 NH1 Argll8 NH2 Argll8 NH1 Arg371 NH2 Arg371 OH Tyr405 NH1 Argll8 OD1 Aspl5l O HOH516 NH2 Arg 152 NH1 Argll8 NH1 Arg371 O H0H516 NH1 Argll8 NH1 Arg371 O H0H516 O H0H516 NeuNAccx(2-8)NeuNAc * 5.1* 4.3* 45* NeuNAca(2-9)Neu NAc (continued in the next page)

29 (Table 3.7 continued) Substrate Protein Distance (A) Atoms Atoms NeuNAca(2-8)Neu NAc Neu NAca(2-9)Neu NAc 06B NH2 Arg SB NE Arg SB NH1 Arg SB 0 H0H SB 0 H0H DB NZLy B OD1 Asn B NE Arg B NH2 Arg B 0 H0H * Favorable interactions, may lead to hydrogen bond formation.

30 Table 3.8 Conformations of the substrates in bound form: Substrate Xi X2 X3 NeuNAca(2-3)GaI NeuNAca(2-6)GaI NeuNAccL(2-8)NeuNAc NeuNAcc(2-9)NeuNAc

31 4g, ''g' 02-C8-C7-C6, C8-C7-C6-05 and C7-C6-05-N5 (approximately -85 0, 3 0, , , -62 1) in NeuNAca(2-8)NeuNAc and, 141g 02-C9-C8-C7, C9-C8-C7-C6 and C8-C7-C6-06 (approximately -73 0, -49 0, 165 0, 176 0, -41 ) in NeuNAca(2-9)NeuNAc favor approximately similar values. In other words, when the first residue (a-neunac) of the SOS fragments are superimposed then the fragment 02-C3-C4-05-C6-06 of Gal of NeuNAca(2-3)Gal, 02-C6-05-C4-C3-H3 of Gal of NeuNAca(2-6)Gal, 02-C8-C7-C6-05-N5 of NeuNAc of NeuNAca(2-8)NeuNAc, 02-C9-C8-C7-C6-06 of NeuNAc of NeuNAca(2-9)NeuNAc can take up similar orientations (Figure 2.16). It may be due to this conformational similarity that the influenza virus N9 neuraminidase enzyme is able to cleave a-neunac residue from the SOS differing in the type of linkages. It is also interesting to note that the electronegative oxygen atoms 04, 05 of the galactose residue of NeuNAca(2-3)Gal, 05, 04 of the galactose residue of NeuNAca(2-6)Gal, 07, 06 of the second NeuNAc residue of NeuNAca(2-8)NeuNAc and 08, 07 of the second NeuNAc residue of NeuNAcct(2-9)NeuNAc also assume similar positions. It is observed that the first set of atoms mentioned in the above substrates are involved in hydrogen bond formation with a water molecule (H0H516) at the active site (Table 3.5 and 3.7). The second sets of atoms are at a distance of about 6 A 77

32 from the side chain electronegative atoms of Arg152 and Arg371, and hence indicating a possibility that water can mediate a hydrogen bond formation. In the disaccharide fragments NeuNAca(2-3)Gal, NeuNAccx(2-8)NeuNAc and NeuNAca(2-9)NeuNAc, the last superimposed atoms are electronegative. These atoms are about 5 A away from the H0H525 (water). It is also of interest to observe that in NeuNAcct(2-3)Gal the 02 of the galactose residue (fixed because of ring geometry) and the 09 of the second NeuNAc residue in NeuNAca(2-8)NeuNAc can also occupy similar positions. These two atoms are having hydrogen bond interaction with the NH2 of the residue Arg152 at the active site (Table 3.5 and 3.7). No corresponding electronegative atoms are present in that position in NeuNAca(2-6)Gal and in NeuNAca(2-9)NeuNAc. This can also attribute for the variation in the enzyme kinetics. When the substrates favours the conformational similarity, the vicinal location of the conserved water position discussed in chapter 2 is occupied by Aspl5l. Hence one can have a notion that these conformational similarities leading to structural similarities may be an essential requirement for the enzyme influenza virus N9 neuraminidase to cleave the SA irrespective of the type of linkages. A schematic representation indicating the conformational preference leading to structural similarity for these substrates was shown in Figure Based on hard sphere energy calculations on the isolated substrates NeuNAca(2-3)Gal, NeuNAca(2-6)Gal and NeuNAca(2-8)NeuNAc, this type of similarity has been proposed earlier (Veluraja and Rao, 1984), the time at 78

33 which the three-dimensional structure of neuraminidases are not known. It is interesting to note that the present study which have been carried out by incorporating the substrates at the active site of neuraminidase still maintains the structural similarity in the bound conformation, an essential requirement for the activity. In order to understand the dynamics of the substrates at the active site, the modeled neuraminidase-sos complexes were further subjected to MD simulations Molecular dynamics simulations of the neuraminidase-sos complexes Since we are interested about the conformational features of the substrates at the active site, it was presumed to perform a 50 Ps MD simulation. The MD generated average structures of neuraminidase-neunaca(2-3)gal, NeuNAcct(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)Neu NAc complexes and the respective starting structures had an RMS deviation of 0.13 A, 0.09 A, 0.14 A and 0.15 A on Ca atoms, 0.16 A, 0.11 A, 0.17 A and 0.17 A for backbone atoms of the neuraminidase respectively. This indicates that there was not much variation in the enzyme structure during MD simulations. The flexibility of the substrate molecules arises mainly because of the glycosidic torsion. An analysis on the glycosidic torsions from the frames collected for every 0.5 ps for the substrates are shown in Figure 3.8, 3.9,

34 C B -ci U) C phi (deg) Figure 3.8 The (g, i4jg) plots obtained over the period of 50 Ps simulation for NeuNAccL(2-3)Ga

35 co 0-4 to A A phi (deg) Figure 3.9 The (mg, ji) plots obtained over the period of 50 Ps simulation for NeuNAca(2-6)Gal

36 C -4 txo C phi (deg) Figure 3.10 The (mg, \Ig) plots obtained over the period of 50 Ps simulation for NeuNAca(2-8)NeuNAc

37 and The distribution of conformations always tends to occur in the allowed region B for NeuNAca(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc. This clearly indicates that all substrates assume unique glycosidic torsions at the active site, which paves a way for conformational equivalence leading to partial structural similarity. The graphical analysis of these MD generated substrates clearly indicates that the SA position is almost similar. In the case of the substrate NeuNAca(2-6)Gal, the distribution of (4, 4Ig) '5 of collected frame of structures is more in region A than in region B. It can be inferred from the graphical analysis that when it occurs in region B, the SA acquires position similar to the other dynamic simulated SOS structures. But when it tends to occur in region A, the position of SA is moved about 2A from the active site, which orientates the second residue (Gal), towards a contact free space. Once this happens in the dynamics it is unable to go back to the allowed region B. The hydrogen bonds that maintain similarity are found to be lost (i.e., the amino acid residues nearby the atoms 04B, 03B, 05B are more than 5A apart). Therefore it can be presumed that whenever NeuNAca(2-6)GaI favors the conformational similarity, i.e., occur in region B the enzyme can recognize it and cleaves, otherwise not. 80

38 C) co ago Q) B (n 0 co phi (deg) Figure 3.11 The (g, Wg) plots obtained over the period of 50 Ps simulation for NeuNAccx(2-9)NeuNAc

39 3.4 Conclusion The influenza virus N9 neuraminidase was able to accommodate the sialic acid in a highly restricted spatial orientation in the Eulerian space at the active site. The Eulerian space allowed for sialic acid at the active site is less than 1%. Molecular mechanics and molecular dynamics calculations of neuraminidases with NeuNAca(2-3)Gal, NeuNAccx(2-6)Gal, NeuNAca(2-8)NeuNAc and NeuNAca(2-9)NeuNAc at the active site reveals that the enzyme can accommodate these disaccharide fragments without any stereochemical clash but with good number of hydrogen bonds between the protein atoms and the substrate atoms. The disaccharide at the active site of influenza virus N9 neuraminidase predominantly prefers a common glycosidic conformation. The common glycosidic conformation which leads to a structural similarity between these disaccharide fragments, perhaps be an essential requirement for the neuraminidase enzyme to recognize and cleave sialic acid irrespective of the type of linkage. N.B.: Region A and B in this chapter corresponds to region B and A in the paper published (Veluraja, K., Suresh, MX, Christlet, T.H.T. and Rafi, Z.A. (2001) J. Biomolec. Struct. Dyna., 19, 33-45). 81