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 differen
Trang 1disulfide linkage
Wei-Hong Du1,2,*, Yu-Hong Han3,4,*, Fei-juan Huang1,*, Juan Li2, Cheng-Wu Chi3,4
and Wei-Hai Fang2
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 (C1–C4, C2–C5, C3–C6) 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: fangwh@bnu.edu.cn
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: chi@sunm.shcnc.ac.cn
*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
1H 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
Trang 2the 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 C1–C6, C2–C4 and
C3–C5 [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 (C1–C5, C2–C4,
C3–C6), 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 H2O 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
daN(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
D1 (p.p.m.)
Fig 2 Sequential daN(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 daNconnectivities are shown for residues 1–3 and 5–16 Residue 4 is proline.
Trang 3compared 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˚
dyanawas used to provide hydrogen-bond
informa-tion during the minimizainforma-tion 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 D2O 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.
d: 3.77, 3.61
e: 7.31 f: 7.54
e: 8.67
d: 0.94 ± 0.89
e: 6.88
Table 2 Structural statistics for the family of 20 structures of cono-toxin mr3e.
AMBER energies, kcalÆmol)1
rmsd to mean coordinates
Rachandran statistics from PROCHECK - NMR
Trang 4The 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 (C1–C5, C2–C4, C3–C6) [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.
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.
Trang 5chains 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]
column The final product was coapplied with native
identity
NMR experiments Samples for NMR experiments were prepared at a
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
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
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
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
backbone amide protons, the sample lyophilized from a
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
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.
Trang 6couplings, was generated from the data and used as input
for dyana (V.1.5) Six u dihedral angles were determined on
of a high resolution 1D proton spectrum of conotoxin mr3e
< 5.5 Hz (Cys3, Phe5, Leu11, Cys12) Backbone dihedral
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
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
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
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