Solution structure of long neurotoxin NTX-1 from the venom of Naja naja oxiana by 2D-NMR spectroscopy Mehdi Talebzadeh-Farooji 1 , Mehriar Amininasab 1 , Maryam M. Elmi 1 , Hossein Naderi-Manesh 2 and Mohammad N. Sarbolouki 1 1 Institute of Biochemistry & Biophysics, University of Tehran, Iran; 2 Faculty of Science, Tarbiat Modares University, Tehran, Iran The N MR solution structures of NTX-1 (PDB code 1W6B and BMRB 6288), a long neurotoxin isolated from the venom of Naja naja oxiana, and the molecular dynamics simulation of these structures a re reported. Calculations are based on 1114 NOEs, 19 hydrogen bonds, 1 9 d ihedral a ngle restraints and secondary chemical shifts derived from 1 Hto 13 C H SQC spectrum. Similar to other long neurotoxins, the three-finger like structure shows a double and a triple stranded b-sheet as well as some flexible r egions, particularly at the tip of loop II and the C-terminal tail. The solution NMR and molecular dynamics s imulated structures are in good agreement with root mean square deviation values of 0.23 and 1 A ˚ for residues involved in b-sheet regions, respectively. The overall fold in the NMR structure is similar to that of the X-ray crystallography, although some differ- ences exist in loop I and the tip of loop II. The most func- tionally important residues are located at the tip of loop II and it appears that the mobility and the l ocal structure in this region modulate the bin ding of NTX-1 and other long neurotoxins to the nicotinic acetylcholine receptor. Keywords: 2D-NMR; long neurotoxins; neurotoxin-1; solution structure; three-finger peptide. Neurotoxins belonging to Elapidae family are divided [1–3] into two groups of short (60–62 amino acids in length with four disulfide bridges) and long neurotoxins (66–74 amino acids with five disulfide bridges). They cause postsynaptic blockage of the nicotinic acetylcholine receptor (AChR) [4,5]. Despite their functional similarity, t hese peptides have differences in their amino acid sequences such as the deletion/insertion of some residues a nd the presence of longer C-ter minal tails in long neurotoxins (Fig. 1). It is also known that the association of long neurotoxins with their receptor, and a lso their dissociation, occurs more slow ly than short neurotoxins [1]. The binding site of neurotoxins is located at the AChR a-subunit between residues 173 and 204, and probably encompasses the agonist-binding site [6]. Because the binding affinity of neurotoxin s to the target receptor is higher than that of acetylcholine, they can completely prevent a cetylcholine b inding [7]. Previous reports have suggested that residues Trp27, Lys25, Arg35, Lys37 and His67 play an important role in receptor b inding and hence in the toxicity of neurotoxins [1,2,6,8]. Already the crystal and solution structures of some long neurotoxins have been reported [9–16]. They usually have a three-finger like structure emerging from a core (globular head) with three loops wherein l oop II plays a key role in binding to AChR. Most of the functionally invariant residues are located here [17]. Basus et al. studied the complex between a-bungarotoxin (from Bungarus multi- cinctus) and the nicotinic receptor peptide by 2D-NMR spectroscopy [6] and reported some differences between the bound and unbound conformations of this neurotoxin particularly at the tip portion of l oop II. Comparison of the crystal and solution structures of a-cobratoxin (a long neurotoxin from Naja naja siamensis, which has 63% homology with NTX-1) revealed that the solution structure is more flexible in the tip of loop II [14]. Here we report the elucidation of solution structure of neurotoxin-1 (NTX-1; P01382, from Naja naja oxiana)via two-dimensional 1 H-NMR spectroscopy. NTX-1, a long neurotoxin with 73 amino acids, is one of the lethal components of Naja naja oxiana venom. This p eptide is different from other neurotoxins as it lacks a phenylalanine residue and has a lower net positive charge [18]. The NMR structure of NTX-1 is compared with the X-ray structure of PDB entry 1NT N [12] whose amino acid sequence d iffers in two positions: deletion of Pro9, and substitution of Asp63 with Asn. Materials and methods Sample preparation and purification Central Asian cobra ( Naja naja oxiana) s nakes were collected, m ilked and the pooled venom lyophilized and Correspondence to H. Nade ri-Manesh, Faculty o f S cience, Tr abiat Modares University, Tehran, Iran, PO Box 14115-175. Fax/Tel.: +98 218 009730, E-mail: naderman@modares.ac.ir and M. N. Sarbolouki, Institute o f Biochemistry & Biophysics, University of Tehran, Tehran, Iran, PO Box 13145-1384. Fax: +98 216 404680, Tel.: +98 216 491267, E-mail: sarbo l@ibb.ut.ac.ir Abbreviations: AChR, nicotinic acetylcholine receptor; a-BTX, a-bungarotoxin; a-CTX, a-cobratoxin; ESI, electron spray ionization; HSQC, heteronuclear single quantum coherence; MD, molecular dynamics; RMSD, root mean square deviation; RP-HPLC, reverse phase high performance liquid chromatography; TPPI, time proportional phase incrementation. (Received 26 June 2004, revised 13 October 2004, accepted 27 October 2004) Eur. J. Biochem. 271, 4950–4957 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04465.x stored at )20 °C. Purification of NTX-1 was performed by the successive application of gel filtration chromatography and R P-HPLC metho ds. The purity, molecular mass, and correspondence with SWISS-PROT code P01382 of NTX-1 were confirmed by electron spray ionization/mass spectro- metry (ESI-MS). NMR spectroscopy NTX-1 was dissolved in D 2 O/H 2 O (10 : 90, v/v) pH 3.2 or 99.9% (v/v) D 2 O to the final concentration of 4 m M .All NMR spectra were recorded on a B ruker DRX-500 spectrometer. Water suppression was achieved by applying WATERGATE pulse sequence. Two-dimensional 1 H- NMR spectra were acquired at 2 0 and 37 °Ctoovercome possible ambiguities in assignments. The spectral width was adjusted to 12.061 p.p.m. (6009.62 Hz) in both dimensions. The carrier freque ncy was set with respect to the c enter of residual water signal and 2,2-dimethyl-2-silapentane-5- sulfonate was used as an internal reference. The time proportional phase incrementation (TPPI) and States-TPPI methods for quadrature detection were used for NOESY [19,20]/TOCSY [19–21] and D QF-COSY [19,20,22] s pectra, respectively. NOESY and TOCSY spectra were acquired at 50, 100, 150 and 200 ms mixing times with 5 6 s cans per each t 1 increment, and 50, 75 and 150 ms mixing times with 48 scans per each t 1 increment, respectively. The DQF-COSY spectrum was acquired in phase sensitive mode with 184 scans per eac h t 1 increment. The spectra were collected using 512 and 2048 complex points in t 1 and t 2 dimensions. Zero filling was performed in t 1 dimension to obtain a final matrix of 1024 · 2048 data points. A natural abundance 1 H- 13 CHSQCspectrumwas acquired at 20 °C with 200 scans p er each t 1 increment. The spectral widths in F1 and F2 dimensions were adjus- ted to 165 .66 p.p.m. (20 833.33 Hz) and 13.33 3 p.p.m. (6666.67 Hz), respectively. All spectra were processed by XWIN - NMR software and analyzed with XEASY [23]. Distance restraints Measuring t he cross-peak intensities from the 150 ms mixing time NOESY spectrum resulted in interproton restraints for NTX-1. The results of hydrogen–deuterium exchange experiments along with preliminary NOE based calculated structures were used to deduce hydrogen bond restraints which were assigned to 2.8 ± 0.5 A ˚ for r N-O and 1.8 ± 0.5 A ˚ for r H–O . / Angle restraints 3 J NH a constants were determined from 2D NOESY spec- trum using the program INFIT [24] and / dihedral angles were restrained to the ranges o f )120° ±30° for 3 J NH a > 8Hzand)60° ±30° for 3 J NH a <6Hz. Secondary structure restraints These were predicted from the observed 13 C a , 13 C b and H a resonances and the use of the chemical shift indexing [25] method along with the TALOS program [26]. Structure calculations Three-dimensional structure calculations were carried out on a P C w orkstation using CNS [27] and the standard protocol o f ARIA 1.1 p rogram [28] under R ed Hat Linux 8.0. A simulated annealing protocol in torsion angle space was used starting from an extended c onformation. The p rotocol was implemented in four stages: (a) high temperature simulated annealing at 10 000 K; (b) a first cooling phase from 10 000 K to 1000 K in 5000 steps; (c) a second cooling phase f rom 1000 K to 5 0 K in 4000 st eps; and (d) 4000 steps refinement and energy minimization. The time s tep f or integration was set to 0.003 ps. One hundred structures were generated in each iteration, and the 20 lowest energy structures in the final iteration were evaluated throu gh PROCHECK [29] and used for further analysis. The structural models were visualized with the program MOLMOL [30]. Molecular dynamics simulations Molecular dynamics (MD) simulations were performed on a PC workstation using the SANDER module of AMBER 7.0 [31] under Red Hat Linux 8.0. All of the calculations were carried out in explicit water with a solvent box whose ed ges were 8 A ˚ apart f rom the closest protein atoms. The fi ve best ARIA structures were first energy minimized in 5000 steps, subjected to 100 ps (2 fs time s teps) of M D at co nstant volume and then gradually heating up from 200 K to 300 K , followed b y 100 ps (2 fs time steps and pressure relaxation time of 2 ps) of MD at 300 K and constant pressure. Finally, 5 00 ps MD simulation with 2 fs time steps and 2 ps pressure r elaxation time a t 300 K were carried out. Results and Discussion Resonance assignment NMR assignments were carried out according to standard methods [32]. Identification of spin systems was made using the DQF-COSY and TOCSY spectra recorded at 20 °C. For assignment of the H a cross-peaks in the region of water suppression, the s pectra obtained at a higher temperature, 37 °C, were used wherein the water s ignal was shifted relative to H a resonances. The results of the spin system identification were confirmed by the 1 H- 13 CHSQCspec- trum. The chemical shifts table of 13 C-NMR for C a and C b and 1 H-NMR has been deposited in BioMagResBank (BMRB), code 6288 (Table S1). To perform the sequential Fig. 1. Multiple sequence alignment o f NTX-1. NTX-1 is aligned with a number of typical long and short neurotoxins, whose three- dimensional structures have been discovered. Ó FEBS 2004 NTX-1 solution structure from N. naja oxiana (Eur. J. Biochem. 271) 4951 assignment, the identified spin systems were sequentially connected by observing H a -H N ,H N -H N and H b -H N cross- peak NOEs in 150 ms NOESY spectrum. Two s uccessive Ala residues, Ala44 and Ala45, and also Trp27 and Trp31 were used as the starting points. The pattern of sequential NOEs for prolines showed that they are in trans confor- mation. Recognition of the secondary structural elements was carried out using long range NOEs between H a protons, protection of H N s in deuterium exchange experi- ments and the secondary chemical shifts from HSQC spectrum. These results indicated the presence of a double- and a triple-stranded antiparallel b-sheet. Structure calculations were based mainly on the NOE restraints, with its sequence distribution being shown in Fig. 2, and other restraints given in the Table 1. On the basis of the total energy, the best 20 refined structures were selected for further analysis and deposited in RCSB protein data bank, PDB code 1W6B. The superimposed structures are displayed in Fig. 3 and their geometric statistics and energetics are summarized in Table 1. All structures are in good agreement with the experimental restraints, none having NOE violations greater than 0.2 A ˚ .The PROCHECK analysis of 20 best st ructures indicates that 97% of nonglycine and nonproline residues lay in the most favored and allowed regions of the Ramachandran plot, while only 0.3% residues are in the disallowed regions (Table 1). Structure description The three-dimensional structure shows a globular head with the emergence of three-finger like l oops. The loop s are cross- linked together by f our disulfide bridges, Cys3-Cys22, Cys15-Cys43, Cys47-Cys58 and Cys59-Cy64, and are involved in a double- and a triple-stranded b-sheet, i.e. the main regular secondary structures of NTX-1. Loop I (residues 1–15) constitutes a double-stranded antiparallel b-sheet (residues 2–5 and 11–14) as well as a linker segment consisting of residues 6–10. This double-stranded b-sheet is stabilized by three hydrogen bond s. The largest loop of NTX-1 (loop II) consists of residues 21–44, w hich is cross-linked to loop I by a disulfide bond between Cys3 and Cys22. The segment connecting loop I and loop II forms a type II b-turn by Ala16-Pro17-Gly18-Gln19 residues. Segments 21 –27 and 38–44 of loop II along with segment 55–59 of loop III form a triple-stranded antiparallel b-sheet held together via h ydrogen bonds. Loop II has a bulky tip with an extra disulfide bridge, Cys28-Cys32, which is not present in short neurotoxins. Although t he tip portion of loop II is poor in medium and long range NOEs (Fig. 2), it shows a local structure like an a-helix in the segments containing residues 3 2–36, as r evealed by H a 32-H N 35 and H a 32-H N 36 NOEs. Loop III, spanning residues 45–59, has two legs, one of which participates in the third strand of the triple stranded b-sheet while the second one having no involvement i n regular secondary elements shows a relatively fixed structure as revealed by the low root mean square deviation (RMSD) in this segment. A disulfide bond between Cys47 and Cys58 stabilizes this loop and a type I turn formed by residues 51–54 connects its two legs. The C-terminal tail of NTX-1 is connecte d to loop III via another turn-like segment containing residues 60–63 and is tethered to it by Cys59-Cys64. This region consists of two distinct segments; the first one, residues 64–68, has a more defined structure in contrast to the second, residues 69–73. It seems that the presence of the disulfide bond and the two proline residues (Pro66 and Pro68) lead to lower mobility of this s egment. In a ddition many long range NOEs between the main chain as well as the side chains of Asn65, Pro66, His67, Pro68 and some residues in loop I and loop II (such as Tyr4, Val38, Ile39, Glu40 and Leu41), show that this region is in close contact with the other parts of the structure. Most of the calculated structures reveal a one-turn a-helix in this region as is confirmed by the observed medium range N OEs. This local structure is not observed in other long neurotoxins. On the other hand, the absence of any NOE between residues 69–73 and other regions of the peptide indicates that there is no s tructural involvement in this region. In contrast to the C-terminal tail, the N-terminal segment has a relatively more defined structure: both Ile1 and Thr2 are engaged in hydrogen bonding, but it seems that Cys15 is not a part of the b-sheet in loop I. The peptide structure has a convex and a concave face (Fig. 4); the l atter being instrumental in receptor binding [2]. The side chains of Leu21, Tyr23, Lys25, Ala44, Pro48 and Ile56 constitute a h ydrophobic cluster on the concave face o f Fig. 2. The number of NOEs per residue used in final structure calculation. Intra residue, sequential, medium range and-long range NOEs are d epicted a s h atched , white, g rey a nd black filled bars, respectively. 4952 M. Talebzadeh-Farooji et al.(Eur. J. Biochem. 271) Ó FEBS 2004 the m olecule, which is postulated to be involved in stability and maintenance of the whole structure [2]. Almost all of these residues are conserved in long neurotoxins (Fig. 1). The side c hain of Tyr23 extends o ver Gly42 o n the neighboring strand and therefore its H N is affected by the ring current of the aromatic group leading to an up-field resonance frequency. Molecular dynamics The structures resulting from MD simulations agree satis- factorily with those of solution structure ensemble. The RMSD for the back bone atoms between the starting and the solvated-minimized structures is about 0.7 A ˚ .During the 500 ps of trajectory used for analysis, the structures were nearly stable and the RMSD alon g t he trajectory was about 3A ˚ for the backbone atoms relative to the initial structure and reduced to 1 A ˚ when only regular secondary elements were regarded. In Fig. 5 parameters such as B-factor, RMSD an d atomic fluctuation per residue for the X-ray and NMR structures and that obtained along the MD trajectory a re shown. In this figure the free C-terminal tail (residues 69–73) has been omitted due to its high level of fluctuation. The limited experimental restraints for the tip portion of loops I, II and III (especially loop II) is consistent with the moderate level of fluctuation a long the MD trajectory. As the tip of loop II is involved in binding to the target receptor it seems t hat flexibility of this region plays a crucial role in adopting the correct conformation. However the possession of residual structure and flexibility for the residues at the tip portion of loop II are supportive of rigid body motion [13] in the way that Gly36 serves as a hinge. Comparison with X-ray structure In Fig. 6C the crystal and the average of NMR structures are superimposed over the backbone atoms. The RMSD for the backbon e atoms of the b-sheet part is 0.72 A ˚ , indicating nearly identical conformation for the solution and c rystal state in this region. The secondary elements of the X-ray structure can be observed in the NMR structures but the length of the b-strand in loop I in solution is somewhat shorter than that of the crystal one (Fig. 6A,B). The presence of an additional p roline in position 9 in NTX-1 (P01382) makes loop I bulkier and more rigid than the corresponding loop in the X-ray structure. It is interesting to note that in contrast to the crystal structure there is n o hydrogen bond network at the tip of loop II, thus residues in this region experience a fast hydrogen–deuterium exchange in D 2 O. The structure calculations show a tendency towards local a-h elix formation (similar to the corresponding X-ray structure) whereas flexibility of the tip of loop II leads to the largest difference between t he two structures in this region. For example, the side chain of Arg35 is in proximity to Trp31 in the crystal structure, whereas the solution structure shows that the aromatic side chains of Trp27 and Trp31 are close together (Fig. 6). The segments connecting the two legs of loop III (type I b-turn) as well as the s egment intervening the loop I and I I (type II b-turn) have a similar conformation in both structures. However, the regular turn elements seen at the tip portion of loop I and residues 60–63 in the crystal structure are not observed in the solution struc- ture. Comparison of crystallographic B factor and RMSD of the NMR s tructures reveals a satisfactory agreement between the two structures (Fig. 5), with both having comparable mobility in the same structural regions. Comparison with other neurotoxins NTX-1 has a significant sequence homology with the following long neurotoxins: toxin b [33], a-BTX [34], a-cobratoxin [35] and LSIII [3 6] with 67%, 63%, 63% Table 1. Structural statistics for ensemble of 20 best structures. Parameter Value Distance restraints Intra residue (i-j ¼ 0) 540 Sequential (|i-j| ¼ 1) 252 Medium range (|i-j| < 5) 39 Long range (|i-j| > 5) 283 Hydrogen bonds 19 Total 1133 Dihedral-angle restraints 3 J NHa 19 Secondary chemical shifts 35 Mean RMS dihedral from experimental restraints NOE (A ˚ ) 0.027 ± 0.002 Dihedrals (°) 0.51 ± 0.15 Mean RMS deviation from idealized covalent geometry Bonds (A°) 0.003 ± 0.00017 Angles (°) 0.44 ± 0.014 Improper (°) 0.36 ± 0.013 Mean energy (kcalÆmol )1 ) E NOE 42.84 ± 7.32 E cdih 380.78 ± 6.84 E vdw )607.98 ± 6.95 E bond 8.87 ± 1.04 E improper 11.016 ± 1.88 E angle 58.72 ± 3.65 E elc )2071 ± 51.29 E total )2220.2 ± 50.73 PROCHECK Ramachandran plot analysis for the best 20 structures Most favored region (%) 72.6 Additionally allowed region (%) 24.7 Generously allowed region (%) 2.4 Disallowed region (%) 0.3 Atomic RMS differences (A ˚ ) Back bone Residues 1–73 1.40 Residues 1–27, 38–68 0.35 b-sheets 0.23 Heavy atom Residues 1–73 1.96 Residues 1–27, 38–68 0.65 b-sheets 0.62 Ó FEBS 2004 NTX-1 solution structure from N. naja oxiana (Eur. J. Biochem. 271) 4953 and 59% identity, respectively (Fig. 1). These t oxins all have 10 cysteine residues that play a major role in determining and maintaining their rigid structures. The structural superposition of these toxins reveals that their overall folding is s imilar. They all show a conformational variability at the tip portion of loop II, with the difference being that the length of the b-strands in NTX-1 is longer t han in the others. These neurotoxins have common structural features that in many respects a re analogous to short ones , although the length of loop I is longer in short neurotoxins. TheNMRstructureofNTX-1isclosesttothatofthe a-cobratoxin crystal. The superposition of these two structures shows an RMSD value of 3.00 A ˚ for all backbone atoms which reduces to 1.4 A ˚ when the C-t er- minal tail and the tip of loop II are ignored. Thus there exists an almost parity between the secondary elements in the t wo structures. It appears that among lo ng neurotoxins a-BTX h as some unique structural features, e.g. while the side chains of Trp27, Arg33 a nd Asp29 in these peptides protrude from the concave f ace of the molecule (a common situation), the aromatic side chain of T rp27 in a-BTX occupies the convex side. The limited b-strands in a-BTX also account for i ts higher flexibility [37]. On the other hand, the substitution o f S er in other long neurotoxins with G ly33 in NTX-1 (Fig. 1) at the tip portion of loop II makes this region conformationally more variable. Binding to the nicotinic acetylcholine receptor (AChR) It has been already shown that long neurotoxins are capable of binding to a7-neuronal as well as muscular AChR [38]. The results from mutational a nalysis of a-cobratox in (a-CTX) indicate that replacement of at least eight residues (Arg33, Lys49, Asp27, L ys23, Phe29, Trp25, Arg36 and Phe65) to unrelated ones causes a remarkable decrease in its affinity towards muscular AChR [ 39]. These r esidues, mostly located at the tip portion of loop II, are conserved or conservatively substituted in the majority of long neurotoxins (Fig. 1). Also the binding role of Lys49 to muscular AChR contributes to the importance of loop III whereas similar studies have showed a trivial participation of this loop in attachment to a7-neuronal AChR [40,41]. However the role of positively charged residues in the binding of a-CTX to the muscular receptor is remarkable Fig. 3. The ensemble of 20 s uperimposed structures. A stereoview of the molecules is illustrated; all of the structures are fitted to the best energetic one wh ich has be en indicated as the neon. Region s i nvolved i n t he fo rmation o f b-sheets are indicated in cyan. Fig. 4. The concave (left) and convex face (right) of NTX-1. The distribution of positive, negative and hyd rophobic residu es are shown in blue, red and white, respectively. 4954 M. Talebzadeh-Farooji et al.(Eur. J. Biochem. 271) Ó FEBS 2004 and four of the above mentioned mutation-sensitive residues are cationic. Because AChR is composed of acidic subunits [2] it s eems t hat the receptor has favorable interactions with cationic groups. Thus it may be expected that the substitution of Arg36 and Lys49 in a-CTX with Val38 and Glu51 in NTX-1, respectively, lead to a weaker interaction with muscular AChR and to a lesser e xtent with a7-neuronal AChR. Interestingly, LSIII, which like NTX-1 does not have these two positively charged residues, shows a weaker and more reversible neurotoxic activity [36]. On the other hand Endo et al. [42] have found that the overall net charge of neurotoxins affects the toxin–receptor inter- actions. Particularly, it should be noted that NTX-1 and LSIII have a net charge of +1 in comparison with +5, +4 and +3 for a-CTX, toxin b and a-BTX, respectively, at pH 7. However, this is not the only factor a ffecting the binding of neurotoxins to t heir receptors. As stated earlier the charac- teristic feature o f all long neurotoxins, playing an important role in their function, is fle xibility at the tip portion of loop II. This affects both the kinetics and thermodynamics of their interactions with the receptor [13,43,44] in a manner that lowers the free energy barrier for complex formation and increases the rate of association. Consequently, the presence of Gly33 inste ad o f Ser at the tip of loop II in NTX-1 makes it more flexible than that of other long neurotoxins (a-BTX, a-CTX, toxin b and LSIII) and allows it to have more conformational diversity when searching for a suitable conformer in the binding cleft. On th e other hand a large flexibility at t his region l eads to a higher entropic c ost during binding of NTX-1 to its receptor, thereby lowering the binding affinity [13]. However, the rigid body motion emerging from some local structure at the tip of loop II decreases t he entropic penalty upon the receptor binding. Local s tructures impose a restriction on freedom in this region and l ower the e ntropy difference between t he free and bound forms of the molecule [44]. Furthermore poor experimental restraints and possibly bulkiness of the tip portion of loop II in long neurotoxins compared to short ones, as well as the finding o f Bystrov et al. [ 45], point to the fact t hat this s egment in the former is likely to be more flexible. This provides a possible explan- ation for the lower dissociation constants of long neuro- toxins from nicotinic acetylcholine receptor c ompared to short ones. Fig. 6. Comparison of NTX-1 NMR and 1NTN X-ray structures. (A) and (B) s how the r ibbo n representation o f t he average d NTX-1 NMR and 1NTN X-ray st ructures, respectively. The hydrop hobic side chains alon g with those important for receptor binding are depicted. (C) The superimposition of the average d NTX-1 NMR (blue ) and 1NTN X-ray ( red) structure s over the backb one atoms. Fig. 5. The comparison of the experimental and MD results. Per residue B f acto r of backb one atoms o f crystalline 1NTN (top), the per residue RMS difference from the mean NMR structure (middle) and per residue atomic fluctuations along t he trajectory of the MD simulation for the five of the 20 best NTX-1 NMR structures (bottom) for resi- dues 1–68. Ó FEBS 2004 NTX-1 solution structure from N. naja oxiana (Eur. J. Biochem. 271) 4955 Conclusions It is concluded that although this group of structures has a typical three-finger shape, they have subtle differences w ith each other. The NMR structure reveals some minor conformational changes that might be very important in studying the mech anism o f t heir receptor binding. The importance of cationic residues in direct b inding of a-CTX to muscular as well as a7-neuronal AChRs has been demonstrated previously [39–41], and thus mutations lead- ing to the removal of the positive charge at Lys25, Arg35 and Lys37 in NTX-1 m ay support this suggestion. Addi- tionally, the substitution of Val38 and Glu51 with positively charged side chains (such as Arg and Lys, similar to the corresponding residues in a-CTX that are involved in receptor binding) would be i n teresting because of the resultant alteration in the binding affinity. Considering flexibility of l oop II in receptor binding, substitution of Gly33 with Ser (a common residue in other long neurotox- ins) may lower mobility, thereby affecting the binding affinity of NTX-1. Acknowledgements The authors w ish t o a cknowledge a nd appreciate the financial s upport by Tehran University’s Research Counc il in the cou rse of this researc h project. They a lso express t heir sincere thanks to Mr M. 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In Toxins as Tools in Neurochemistry (Hucho, F. & Ovc hinnikov, Y.A., eds), pp. 193–233. Proceeding of the Symposium held at Berlin, 1983, Walter de Gruyter, Berlin. Supplementary material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4465/EJB4465sm.htm Table S1. The c hemical shift table of NTX-1. Ó FEBS 2004 NTX-1 solution structure from N. naja oxiana (Eur. J. Biochem. 271) 4957 . Solution structure of long neurotoxin NTX-1 from the venom of Naja naja oxiana by 2D-NMR spectroscopy Mehdi Talebzadeh-Farooji 1 ,. of NTX-1 (PDB code 1W6B and BMRB 6288), a long neurotoxin isolated from the venom of Naja naja oxiana, and the molecular dynamics simulation of these structures