Báo cáo khoa học: Solution structure of an M-1 conotoxin with a novel disulfide linkage pdf

Solution structure of an M-1 conotoxin with a novel
disulfide linkage
Wei-Hong Du
1,2,
*, Yu-Hong Han
3,4,
*, Fei-juan Huang
1,
*, Juan Li
2
, Cheng-Wu Chi
3,4
and Wei-Hai Fang
2
1 Department of Chemistry, Renmin University of China, Beijing, China
2 Department of Chemistry, Beijing Normal University, China
3 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School
of the Chinese Academy of Sciences, Shanghai, China
4 Institute of Protein Research, Tongji University, Shanghai, China
Over their 50 million years of evolution, cone snails
have developed a series of small disulfide-rich peptides
(conotoxins) in their venoms. Each peptide can selec-
tively target a specific isoform of ion channel or mem-
brane receptor [1,2]. Although it is estimated that each
species of cone snail possesses 50–200 conotoxins in its
arsenal, and there are more than 50 000 known cono-
toxins, the majority belong to several gene super-
families and only several structural motifs are widely
shared.
M-superfamily conotoxins form a group with a typ-
ical cysteine arrangement of (-CC-C-C-CC-) and par-

ticularly highly conserved signal peptide sequences.
Depending on the number of residues located in the
last cysteine loop, the M-superfamily has been provi-
sionally divided into four branches, M-1, M-2, M-3
and M-4 [3]. The M-superfamily conotoxins l-, w- and
jM-conotoxins (22–24 amino acids) have all been iden-
tified from fish-hunting cone snails and belong to M-4
branch [4–6]. Although they have diverse molecular
targets (Na
+
channel, nicotinic acetylcholine receptor
and K
+
channel, respectively), they share a disulfide
connectivity (C
1
–C
4
,C
2
–C
5
,C
3
–C
6
) and common
backbone scaffold [7]. In contrast to the M-4 branch,
Keywords
disulfide linkage; M-conotoxin; mr3e; NMR;

solution structure
Correspondence
W H. Fang, Department of Chemistry,
Beijing Normal University, 19 Xin Jie Kou
Wai St., Beijing 100875, China
Fax: +86 10 5880 2075
Tel: +86 10 5880 5382
E-mail:
C W. Chi, Shanghai Institute of
Biochemistry and Cell Biology, Chinese
Academy of Sciences, 320 YueYang Road,
Shanghai 200031, China
Fax: +86 21 5492 1011
Tel: +86 21 5492 1165
E-mail:
*These authors contributed equally to this
study
(Received 10 December 2006, revised 7
March 2007, accepted 16 March 2007)
doi:10.1111/j.1742-4658.2007.05795.x
The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-
CC-), and is one of the eight major superfamilies found in the venom of
the cone snail. Depending on the number of residues located in the last
Cys loop (between Cys4 and Cys5), the M-superfamily family can be divi-
ded into four branches, namely M-1, -2, -3 and -4. Recently, two
M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a
new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4,
and Cys3 and Cys6, which is different from those seen in the M-2 and M-4
branches. Here we report the 3D structure of mr3e determined by 2D
1

H NMR in aqueous solution. Twenty converged structures of this peptide
were obtained on the basis of 190 distance constraints obtained from NOE
connectivities, as well as six u dihedral angle, three hydrogen bond, and
three disulfide bond constraints. The rmsd values about the averaged
coordinates of the backbone atoms were 0.43 ± 0.19 A
˚
. Although mr3e
has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a
distinctive backbone conformation with the overall molecule resembling
a ‘flying bird’. Thus, different disulfide linkages may be employed by
conotoxins with the same Cys framework to result in a more diversified
backbone scaffold.
2596 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS
the other three branches of the M-superfamily are rel-
atively small (12–19 amino acids) and are found mostly
in mollusk- and worm-hunting cone snails. Disulfide
linkage analyses of two M-2 branch conotoxins, mr3a
and tx3c, have shown that they possess a distinctive
disulfide bond arrangement of C
1
–C
6
,C
2
–C
4
and
C
3
–C

5
[3]. In addition, BtIIIB, another M-2 branch
conotoxin from the venom of a vermivorous cone snail
Conus betulinus, has been proven to have the same
disulfide linkage as mr3a and tx3c [8].
Recently, a third disulfide bond arrangement within
M-superfamily conotoxins has been characterized. Two
M-1 branch conotoxins, mr3e (Fig. 1) and tx3a were
found to have a new disulfide linkage (C
1
–C
5
,C
2
–C
4
,
C
3
–C
6
), which differs from those seen in M-2 and M-4
branch conotoxins [9]. Here we report the 3D structure
of mr3e, a novel M-1 branch conotoxin with the above
new disulfide connectivity.
Results
Sequence-specific resonance assignments
2D NMR spectroscopy was used to investigate the 3D
structure of conotoxin mr3e in aqueous solution at
pH 3. Proton resonances for conotoxin mr3e were

assigned using established methods [10]. Fourteen of
the 16 spin systems were found in the ‘fingerprint’
region of a 120 ms TOCSY spectrum. TOCSY assign-
ments for Val1, Pro4, Phe5, His9, Leu11, Tyr13 and
Asp16 were verified in the fingerprint region of a
DQF-COSY spectrum.
The sequential assignments of amino acids in the
primary sequence started with the unique residues
Leu11 and His9. A NOESY ‘walk’ toward the N-ter-
minus identified the residues from Leu11 to His9 and
one toward the C-terminus identified the residues from
Cys12 to Asp16. Two of the glycine spin systems iden-
tified in the TOCSY spectrum were encountered in a
NOESY walk at positions 6 and 7. Residues from
Cys2 to Cys8 were then assigned along the ‘walk’. The
phenylalanine at position 5 was confirmed during the
process. Pro4 was assigned by its TOCSY spin system
and the NOEs from its a-proton to the amide proton
of Phe5, and from its d-proton to the a-proton of
Gly7. The valine residue at position 1 was finally
assigned on the basis of NOEs from the a- and b-pro-
tons of Val1 to the amide proton of Cys2.
The amide proton of Val1 disappeared in the H
2
O
spectrum, possibly because of its special position at the
N-terminus and fast exchange in water. NOESY data
acquired at 294 K for conotoxin mr3e showed a large
number of NOEs which suggested that the structure of
the peptide was sufficiently constrained for distance-

geometry calculations. Figure 2 shows the sequential
d
aN(i,i+1)
connectivities on the CaH-NH fingerprint
region of the NOESY spectrum with a mixing time of
200 ms. All chemical shifts are listed in Table 1.
Structure calculation and evaluation
NMR experiments provided enough distance and angle
constraints to calculate the structure of mr3e. The con-
straints for structure elucidation were determined from
a survey of NMR data using the traditional visual ana-
lysis method developed by Wuthrich [10]. In total, 169
distance constraints were obtained from the 200-ms
NOESY spectrum. Six u angle constraints and three
disulfide bonds from Cys2 to Cys14, Cys3 to Cys12,
and Cys8 to Cys15 were added to the distance
constraints for primary structure determination. A set
of 20 structures was generated with a mean global
rmsd of 1.88 A
˚
using the dyana (v. 1.5) [11] software
package. The lowest energy structure was then dis-
played, and ambiguous NOESY signals were evaluated
Fig. 1. Conotoxin mr3e sequence and its disulfide linkage.
3.5
E10
V1
G6
F5
Y13

L11
C3
D16
C8
C2
C15
G7
C14
C12
H9
4.0
4.5
5.0
9.5 9.0 8.5 8.0 7.5
D1 (p.p.m.)
D2 (p.p.m.)
Fig. 2. Sequential d
aN(i,i+1)
connectivities in the CaH-NH fingerprint
region of the NOESY spectrum. The mixing time for the NOESY
spectrum is 200 ms. Sequential d
aN
connectivities are shown for
residues 1–3 and 5–16. Residue 4 is proline.
W H. Du et al. Solution structure of an M-1 conotoxin
FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2597
compared with those of the partially minimized struc-
ture. Twenty-one distance constraints were added on
the basis of this analysis, and the minimization process
was repeated to generate a set of 15 structures with a

mean global rmsd of 0.69 A
˚
.
dyana was used to provide hydrogen-bond informa-
tion during the minimization. Deuterium-exchange
studies indicated that hydrogen bonds might form
exCysist between the amide protons of Gly7, Cys8 and
Cys12 and nearby oxygen or nitrogen atoms. The reso-
nances of amide protons in these residues were not
diminished after 3 h in D
2
O at 294 K in a 1D proton
time course experiment. dyana provided hydrogen
bond acceptor oxygen and nitrogen atoms for each of
the amide protons from these four residues. The
hydrogen bonds for Gly7, Cys8 and Cys12 were used
as constraints. Thus, six upper and six lower distance
constraints were added for the hydrogen-bond interac-
tions, and another round of minimization was per-
formed. The result was a final set of 20 structures with
a mean global backbone rmsd of 0.56 ± 0.16 A
˚
and a
mean global heavy atom rmsd of 1.30 ± 0.28 A
˚
.
Finally, refinement of the structure was carried out
using amber 5 [12] for energy minimization. An
ensemble of 20 structures with lower energy and better
Ramachandran plots was chosen to represent the 3D

solution fold of conotoxin mr3e and the mean struc-
ture was generated using molmol [13]. The program
procheck was used to analyze the family of 20 struc-
tures [14]. Structural statistics are shown in Table 2.
The 20 structures converged to a common fold; the
rmsd values of 20 structures are low. The coordinates
for the family of 20 structures and NMR constraints
file have been deposited in the Brookhaven Protein
Data Bank (PDB) with accession number 2EFZ.
3D structure of mr3e
Figure 3 shows an overlay of the backbone atoms for
the 20 structures of mr3e. The overall rmsd reported
for the final 20 structures (0.43 ± 0.19 A
˚
) is influenced
by disorder in the C-terminal residue Asp16. When
Asp16 is eliminated and the molecule is minimized by
considering the first 15 residues only, the mean global
backbone rmsd decreases from 0.43 to 0.24 A
˚
. Unlike
the N-terminal portion, the C-terminal portion of the
molecule is poorly resolved.
The refined structure of conotoxin mr3e contains
two turns defined by residues Phe5 to Cys8 and His9
to Cys12 (Fig. 4). The residues from Phe5 to Cys8 are
characteristic of a type I b-turn with a glycine residue
(Gly7) at position i+2. The glycine residue is required
to accommodate the necessary angle constraints of the
turn. The second turn in the region between His9 and

Cys12 is apparently stabilized by a hydrogen bond
between the carbonyl oxygen of His9 and the amide
proton of Cys12. The interaction is characteristic of a
type II b-turn.
Table 1. Proton resonance assignments (p.p.m.) for mr3e.
Residue HN abOther
Val1 3.88 2.25 c: 1.06 ± 1.03
Cys2 8.79 4.92 2.72, 2.51
Cys3 8.49 4.49 3.72, 3.47
Pro4 4.6 2.30, 2.03 c: 2.11, 1.81
d: 3.77, 3.61
Phe5 8.75 4.2 3.05, 2.98 d: 7.26
e: 7.31
f: 7.54
Gly6 8.65 4.03, 3.53
Gly7 8.18 4.58, 3.31
Cys8 8.42 4.7 3.06
His9 7.3 4.89 3.53, 3.26 d: 7.29
e: 8.67
Glu10 8.92 4.15 2.13 c: 2.55
Leu11 8.45 4.08 2.01, 1.72 c: 1.61
d: 0.94 ± 0.89
Cys12 7.57 4.46 3.22, 3.17
Tyr13 9.08 4.34 3.23, 3.04 d: 7.17
e: 6.88
Cys14 7.96 4.64 3.79, 3.16
Cys15 9.44 5.07 3.42, 3.03
Asp16 8.76 4.49 2.69, 2.52
Table 2. Structural statistics for the family of 20 structures of cono-
toxin mr3e.

Experimental constraints Number
Intraresidual 122
Sequential (|I ) j | ¼ 1) 48
Medium range 7
Long range 13
AMBER energies, kcalÆmol
)1
Bond 2.822 ± 0.136
Angle 34.311 ± 1.454
Dihedral 36.114 ± 2.807
VDW )55.829 ± 1.478
EEL )442.969 ± 13.886
H-bond )4.178 ± 1.079
Constraints 2.551 ± 0.438
Total )98.210 ± 8.976
rmsd to mean coordinates
Backbone atoms 0.43 ± 0.19 A
˚
Nonhydrogen heavy atoms: 1.26 ± 0.30 A
˚
Rachandran statistics from
PROCHECK-NMR
Most favored regions, % 78.6
Additional allowed regions, % 14.1
Generously allowed regions, % 7.3
Disallowed regions, % 0
Solution structure of an M-1 conotoxin W H. Du et al.
2598 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS
The 3D structure of mr3e is well defined. Figure 5
shows the backbone structure along with front, side and

back views of the surface of the peptide. The double-
turn conformation in conotoxin mr3e produces an over-
all shape of a ‘flying bird’ when viewed from the front.
Discussion
M-superfamily conotoxins, one of the major groups of
disulfide-rich peptides, are widely distributed in the
venoms of all three feeding types of cone snails.
Depending on the number of residues located in the
last Cys loop, M-superfamily conotoxins have been
provisionally divided into four branches, namely M-1,
-2, -3, -4. Interestingly, to the best of our knowledge,
three different disulfide linkages can be found in
M-1 (1–5, 2–4, 3–6), M-2 (1–6, 2–4, 3–5) and M-4
(1–4, 2–5, 3–6) branch conotoxins, respectively.
mr3e is an M-1 branch conotoxin purified from the
venom of a mollusk-hunting cone snail, C. marmoreus;
it has 16 amino acids in its mature peptide. Previously,
we have shown that mr3e is characterized by its dis-
tinctive disulfide connectivity (C
1
–C
5
,C
2
–C
4
,C
3
–C
6

)
[9], which is completely different from those of well-
studied l-, w- and jM-conotoxins (M-4 branch) and
the recently reported, comparatively small, excitory
M-superfamily conotoxins mr3a, mr3b and tx3c (M-2
branch). In this report, we show that there are two
classic b-turns involved in the tertiary structure of
mr3e (Fig. 6A). The backbone conformation of mr3e
is different from that of the M-2 branch conotoxin
mr3a, which possesses a distinctive triple-turn back-
bone structure motif (Fig. 6B) [15]. Such a triple-turn
motif makes the mr3a molecule fold into a tight and
globular structure (Fig. 6D). By contrast, the double-
turn motif of mr3e, which apparently results from its
differing disulfide bond arrangement, gives the mr3e a
more irregular overall molecular shape, with the side
Fig. 4. Backbone peptide folding of mr3e. Turn 1 between Phe5
and Cys8 and turn 2 between His9 and Cys12 are shown in green.
Fig. 3. Overlay of the backbone atoms for the 20 converged struc-
tures of conotoxin mr3e. The C-terminal Asp is seen to be in a
poorly resolved region of the molecule.
Backbone Front
Back Side
Fig. 5. 3D structure of mr3e. The backbone structure is shown
along with front, back, and side views of the surface of M-1 branch
conotoxin mr3e. Blue regions are hydrophobic, and red regions are
hydrophilic.
W H. Du et al. Solution structure of an M-1 conotoxin
FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2599
chains of several amino acids protruding outside the

molecule (Fig. 6C).
In contrast to the typical excitory symptoms, such
as circular movements, barrel rolling and convulsions,
elicited by cranial injection of mr3a [3], mr3e has no
obvious effect on mice [9]. Therefore, it is most likely
that these two conotoxins have different physiological
functions, and this is not surprising considering that
they have completely different backbone scaffolds.
Although M-1 and M-2 branch conotoxins are similar
in size and cysteine framework, and are all abundant
in mollusk- and worm-hunting cone snails, more evi-
dence has emerged that they are phylogenetically diver-
gent groups. These two groups of M-superfamily
conotoxins differ with respect to signal peptide
sequence, disulfide linkage, backbone scaffold and
most likely molecular target.
It seems to be a favored strategy of cone snails to
generate different backbone scaffolds within conotox-
ins by introducing different disulfide linkages into
conotoxins that share the same cysteine framework.
For instance, a-conotoxin and v-conotoxin share the
same ‘-CC-C-C-’ cysteine framework, but differ greatly
in disulfide linkage, backbone scaffold and conse-
quently molecular target [16–18]. Such a strategy,
which yields more structural and functional diversity
in the conotoxins, will help cone snails to survive
severe environmental pressures.
Experimental procedures
Peptide synthesis and refolding
mr3e was chemically synthesized as described previously [9].

Linear peptide was oxidized by air in 50 mm NH
4
HCO
3
buffer and purified on a semi-preparative C
18
reverse-phase
column. The final product was coapplied with native
mr3e to an analytical C
18
reverse-phase column to verify its
identity.
NMR experiments
Samples for NMR experiments were prepared at a concentra-
tion of  2.0 mm in either 99.99% D
2
O (Cambridge Isotopes,
Andover, MA, USA) or 9 : 1(v ⁄ v) H
2
O ⁄ D
2
O with 0.01%
trifluoroacetic acid, at pH 3.0. NMR measurements were
performed using standard pulse sequences and phase cycling
on a Bruker Avance 500 NMR spectrometer at 294 K.
Proton DQF-COSY, NOESY and TOCSY spectra in
99.99% D
2
O and in 9 : 1 H
2

O ⁄ D
2
O(v⁄ v) were acquired
with the transmitter set at 4.80 p.p.m. and a spectral win-
dow of 6000 Hz. All 2D NMR spectra were acquired in a
phase-sensitive mode using time-proportional phase incre-
mentation for quadrature detection in the t
1
dimension.
Presaturation during the relaxation delay period was used
to solvent resonance. A series of NOESY spectra was
acquired with mixing times of 400, 200, 150 100 and 50 ms.
TOCSY spectra under both solvent conditions were
acquired with a mixing time of 120 ms.
Spectra were processed using xwinnmr or topspin soft-
ware. Phase-shifted sine-squared window functions were
applied before Fourier transformation, with shifts of 60 or
90 ° in both dimensions. Final matrix sizes were usually
2048 · 2048 real points. To identify the slow exchange of
backbone amide protons, the sample lyophilized from a
H
2
O solution was redissolved in D
2
O. 1D
1
H spectra were
measured after 5 min, and subsequently every 0.5 h up to
20 h. Chemical shifts were referenced to the methyl reson-
ance of 4,4-dimethyl-4-silapentane-1-sulfonic acid used as

an internal standard.
Distance restraints and structure calculations
An initial survey of distance constraints was performed on a
series of NOESY spectra acquired at mixing times of 400,
200, 150, 100 and 50 ms. Build-up curves were produced
which showed a leveling of the intensity of the NOE at mix-
ing times > 200 ms. Quantitative determination of the
cross-peak intensities was based on counting the contour
levels. Off-diagonal resonances were classified as strong,
medium or weak on the basis of their relative intensities and
set to distance constraints of 1.8–2.5, 1.8–3.5, and 1.8–5.5 A
˚
,
respectively. A set of 96 intra- and interproton distance
restraints, representing unambiguously assigned dipolar
AB
CD
Fig. 6. Comparison of 3D structure of mr3e (M-1 branch conotoxin)
and mr3a (M-2 branch conotoxin). (A,B) Backbone conformation of
mr3e and mr3a. (C,D) Surface representation of mr3e and mr3a.
Blue regions are hydrophobic, and red regions are hydrophilic.
Solution structure of an M-1 conotoxin W H. Du et al.
2600 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS
couplings, was generated from the data and used as input
for dyana (V.1.5). Six u dihedral angles were determined on
the basis of the
3
J
NHa
coupling constants derived by analysis

of a high resolution 1D proton spectrum of conotoxin mr3e.
The u angle constraints were set to )120 ± 40° for
3
J
NHa
> 8.0 Hz (Gly7, Glu10) and to )65±25° for
3
J
NHa
< 5.5 Hz (Cys3, Phe5, Leu11, Cys12). Backbone dihedral
constraints were not applied for
3
J
NHa
values between 5.5
and 8.0 Hz. After the initial calculation, hydrogen-bonds
constraints were added as target values of 2.2 A
˚
for NH(i)–
O(j) and 3.2 A
˚
for N(i)–O(j), respectively.
One thousand random structures were generated by
dyana (v. 1.5) that fit the primary sequence and covalent
and spatial requirements of mr3e. A total of 190 distance
constraints, six u angle restraints and three hydrogen bonds
constraints were input for the molecular modeling protocol
for the dyana algorithm. The outcome was a set of 20
structures with a mean global rmsd of 0.56 ± 0.16 A
˚

and a
mean global heavy atom rmsd of 1.30 ± 0.28 A
˚
.
Structural refinement was carried out using amber 5
and structure quality was analyzed using molmol and
procheck-nmr.
Acknowledgements
This work was supported by the National Basic
Research Program of China (2004CB719900) and the
National Natural Science Foundation of China
(20473013). We thank CD Poulter (Department of
Chemistry, Southern Oregon University) for gener-
ously providing the pdb file of M-2 branch mr3a.
References
1 Olivera BM (1997) E.E. Just lecture, 1996. Conus venom
peptides, receptor and ion channel targets, and drug
design: 50 million years of neuropharmacology. Mol
Biol Cell 8, 2101–2109.
2 Terlau H & Olivera BM (2004) Conus venoms: a rich
source of novel ion channel-targeted peptides. Physiol
Rev 84, 41–68.
3 Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M,
Walker C, Colledge C, Garrett JE, McDougal O, Li W,
Gray WR et al. (2005) Definition of the M-conotoxin
superfamily: characterization of novel peptides from
molluscivorous Conus venoms. Biochemistry 44,
8176–8186.
4 Cruz LJ, Kupryszewski G, LeCheminant GW, Gray
WR, Olivera BM & Rivier J (1989) Mu-conotoxin

GIIIA, a peptide ligand for muscle sodium channels:
chemical synthesis, radiolabeling, and receptor charac-
terization. Biochemistry 28, 3437–3442.
5 Shon KJ, Grilley M, Jacobsen R, Cartier GE, Hopkins
C, Gray WR, Watkins M, Hillyard DR, Rivier J,
Torres J et al. (1997) A noncompetitive peptide
inhibitor of the nicotinic acetylcholine receptor from
Conus purpurascens venom. Biochemistry 36, 9581–
9587.
6 Ferber M, Sporning A, Jeserich G, De La Cruz R,
Watkins M, Olivera BM & Terlau H (2003) A novel
Conus peptide ligand for K
+
channels. J Biol Chem
278, 2177–2183.
7 Al-Sabi A, Lennartz D, Ferber M, Gulyas J, Rivier JE,
Olivera BM, Carlomagno T & Terlau H (2004)
Kappa M-conotoxin RIIIK, structural and functional
novelty in a K
+
channel antagonist. Biochemistry 43,
8625–8635.
8 Zhao TY, Cao Y, Dai XD, Fan CX & Chen JS (2005)
Purification, sequence and disulfide bonding pattern of
a novel conotoxin BtIIIB. Acta Chimica Sinica 63,
163–168.
9 Han YH, Wang Q, Jiang H, Liu L, Xiao C, Yuan DD,
Shao XX, Dai QY, Cheng JS & Chi CW (2006) Charac-
terization of novel M-superfamily conotoxins with new
disulfide linkage. FEBS J 273, 4972–4982.

10 Wu
¨
thrich K (1986) NMR of Proteins and Nucleic Acids.
Wiley, New York.
11 Guntert P, Mumenthaler C & Wuthrich K (1997)
Torsion angle dynamics for NMR structure calculation
with the new program DYANA. J Mol Biol 273,
283–298.
12 Pearlman DA, Case DA, Caldwell DA, Ross WR,
Cheatham TE, DeBolt S, Ferguson D, Seibel G &
Kollman P (1995) AMBER, a computer program for
applying molecular mechanics, normal mode analysis,
molecular dynamics and free energy calculations to
elucidate the structures and energies of molecules.
Comput Phys Commun 91, 1–41.
13 Koradi R, Billeter M & Wuthrich K (1996) MOLMOL:
a program for display and analysis of macromolecular
structures. J Mol Graphics 14(51–55), 29–32.
14 Laskowski RA, Rullmannn JA, MacArthur MW,
Kaptein R & Thornton JM (1996) AQUA and PRO-
CHECK-NMR: programs for checking the quality of
protein structures solved by NMR. J Biomol NMR 8,
477–486.
15 McDougal OM & Poulter CD (2004) Three-dimensional
structure of the mini-M conotoxin mr3a. Biochemistry
43, 425–429.
16 Balaji RA, Ohtake A, Sato K, Gopalakrishnakone P,
Kini RM, Seow KT & Bay BH (2000) Lamda-conotox-
ins, a new family of conotoxins with unique disulfide
pattern and protein folding. Isolation and characteriza-

tion from the venom of Conus marmoreus. J Biol Chem
275, 39516–39522.
17 McIntosh JM, Corpuz GO, Layer RT, Garrett JE,
Wagstaff JD, Bulaj G, Vyazovkina A, Yoshikami D,
Cruz LJ & Olivera BM (2000) Isolation and characteri-
W H. Du et al. Solution structure of an M-1 conotoxin
FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2601
zation of a novel conus peptide with apparent antinoci-
ceptive activity. J Biol Chem 275, 32391–32397.
18 Sharpe IA, Gehrmann J, Loughnan ML, Thomas L,
Adams DA, Atkins A, Palant E, Craik DJ, Adams DJ,
Alewood PF et al. (2001) Two new classes of conopep-
tides inhibit the alpha1-adrenoceptor and noradrenaline
transporter. Nat Neurosci 4, 902–907.
Solution structure of an M-1 conotoxin W H. Du et al.
2602 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS