Báo cáo khoa học: Solution structure of long neurotoxin NTX-1 from the venom of Naja naja oxiana by 2D-NMR spectroscopy pot

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: 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
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. Erfani and t he
NMR fac ility a t T arbia t Mo dares U niversity for their valuable
assistance with NMR spectra .
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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