From the Institute for Molecular Bioscience and
§ School of Biomedical Sciences, University of
Queensland, Brisbane, Queensland 4072, Australia
Received for publication, October 8, 2002
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ABSTRACT |
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Using assay-directed fractionation of
Conus geographus crude venom, we isolated Neuronal nicotinic acetylcholine receptors
(nAChR)1 represent important
targets for the development of novel drugs for the treatment of pain
and various disorders of the central nervous system (1). To date, eight
Conotoxins are small disulfide-rich peptides from the venom of the
predatory marine snails of the genus Conus. These
miniproteins have proved to be valuable tools for investigating the
structure and function of ligand- and voltage-gated ion channels.
-conotoxin
GID, which acts selectively at neuronal nicotinic acetylcholine
receptors (nAChRs). Unlike other neuronally selective
-conotoxins,
-GID has a four amino acid N-terminal tail,
-carboxyglutamate
(Gla), and hydroxyproline (O) residues, and lacks an amidated C
terminus. GID inhibits
7 and
3
2 nAChRs with IC50
values of 5 and 3 nM, respectively and is at least
1000-fold less potent at the
1
1
,
3
4, and
4
4 combinations. GID also potently inhibits the
4
2 subtype
(IC50 of 150 nM). Deletion of the N-terminal
sequence (GID
1-4) significantly decreased activity at the
4
2
nAChR but hardly affected potency at
3
2 and
7 nAChRs, despite
enhancing the off-rates at these receptors. In contrast,
Arg12 contributed to
4
2 and
7 activity but not to
3
2 activity. The three-dimensional structure of GID is well
defined over residues 4-19 with a similar motif to other
-conotoxins. However, despite its influence on activity, the tail
appears to be disordered in solution. Comparison of GID with other
4/7-conotoxins which possess an NN(P/O) motif in loop II,
revealed a correlation between increasing length of the
aliphatic side-chain in position 10 (equivalent to 13 in GID) and
greater
7 versus
3
2 selectivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and three
subunits (
2-
7,
9,
10,
2-
4) of the
nAChRs have been cloned from sensory and neuronal mammalian cells
(2-4). For the
7 and
9 subunits, it has been shown that they
need no additional subunits to form functional ion channels upon
heterologous expression. All other
subunits, however, require at
least the co-expression of one
subunit, or another
subunit in
the case of
10. Ternary combinations of two different
and one
subunit or two different
and one
subunit, and even
quaternary combinations have been described (5, 6). This diversity of
subunit combinations has the potential to generate a wide range of
receptor subtypes with different pharmacological and functional
properties. To help unravel which native neuronal nAChR subunit
combinations are responsible for specific physiological functions,
additional selective inhibitors are required.
-Conotoxins are competitive antagonists of acetylcholine (ACh)
binding to the nAChR (7). The
-conotoxins described so far are among the most selective inhibitors to be identified (Fig.
1). They are typically 12-18 amino acids
long, contain a conserved Pro in loop I, and are folded by two
disulfide bonds connecting Cys1-Cys3 and
Cys2-Cys4. Based on the number of amino acids
between the second and third cysteine residues (loop I) and the third
and fourth cysteine residues (loop II) they are divided into
3/5,
4/7,
4/6, and
4/3 structural subfamilies (8). The
3/5-conotoxins are selective for the muscle-type nAChR, while most
4/7- and
4/6-conotoxins are selective for neuronal nAChRs. An
exception is the
4/7-conotoxin EI, which binds to the
/
interface of the muscle-type nAChR (9). The
4/3-type characterized
by ImI is
7-selective (10). The three-dimensional structures of
different neuronal-specific and muscle-specific
-conotoxins have
been determined by NMR (11-15) and x-ray structural analysis (16-19).
The
4/7-conotoxins and ImI share similar backbone conformations and
a rigid hydrophobic core (14), suggesting that their different
specificities for nAChR subtypes arise from the different amino acid
side-chains projecting from this conserved scaffold.
View larger version (48K):
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Fig. 1.
Comparison of -GID
and related neuronally active
-conotoxins. A, sequence and
selectivity of neuronally active
-conotoxins. A motif (NN(O/P))
commonly found in the second loop of
4/7-conotoxins is marked in
gray. Asterisks indicate C-terminal amidation.
Sulfated Tyr are underlined,
,
-carboxyglutamic acid,
and O, hydroxyproline. B, sequences of GID analogues
investigated in this study. Mutated residues are marked in
gray. Selectivity determined from oocyte studies except for
EpI, which was determined from studies in neurons.
The 4
2 nAChRs together with the
7 subtype are the most
abundant nAChRs in mammalian brain. Knockout studies in mice have revealed an important role for
4 and
2 in pain and cognition (20)
but further investigations of its role are limited by the lack of
4
2-selective inhibitors. A primary goal of this study was to
identify new
-conotoxins active at the
4
2 subtype. In this
report, we describe the isolation and characterization of
4/7-conotoxin GID. GID possesses a novel four amino acid N-terminal tail and an Arg in position 12 that contribute to
4
2 selectivity. The NMR structure of GID was determined to define the location of
structural features that contribute to the selectivity of GID.
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EXPERIMENTAL PROCEDURES |
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Isolation, Purification, and Sequencing of GID
Extraction of Crude Venom--
Nine specimens of
Conus geographus were collected from the Great Barrier Reef,
Australia. Crude venom duct contents were extracted with 30%
acetonitrile/water acidified with 0.1% trifluoroacetic acid and
centrifuged. Soluble material (120 mg) was lyophilized and stored at
20 °C prior to use.
Isolation and Purification of GID-- A portion of the crude venom extract (40 mg) was fractionated on a semipreparative RP-HPLC column (10-µm C18, Vydac) eluted at 3 ml/min with a linear gradient of 0-80% solvent B over 80 min (solvent A, 0.1% trifluoroacetic acid; solvent B, 90% acetonitrile, 0.09% trifluoroacetic acid). Inhibition in a functional nAChR assay (see below) identified an active 1-min fraction eluting at ~21 min (Fig. 2A), which was further purified by RP-HPLC employing a linear gradient of 0-25% B over 25 or 45 min (5-µm C18, 46 × 250 mm Zorbax or Jupiter columns at 1 ml/min or a SB300 3.5 µm C18, 2.1 × 50 mm Zorbax column at 0.2 ml/min eluted with solvent A, 0.05% trifluoroacetic acid; solvent B, 90% acetonitrile and 0.045% trifluoroacetic acid). An active peptide detected at 214 nm was collected and sequenced after reduction and alkylation with maleimide (see below). Venom was further characterized by LC/MS analysis using a PESciex API QSTAR Pulsar mass spectrometer (m/z 500-2200) to monitor the eluant from a 5-µm C3, 2.1 × 150 mm Zorbax column eluted with 0-60% B over 60 min (A, 0.1% formic acid; B, 90% aqueous acetonitrile, 0.09% formic acid).
Sequencing--
The purified peptide (~20 pmol) was reduced in
the presence of 10 mM TCEP and 50 mM ammonium
acetate, pH 4.5 (37 °C for 1 h) before alkylating in the added
presence of 20 mM maleimide (37 °C for 1 h). The
alkylated peptide was repurified by RP-HPLC prior to sequence analysis
by Edman chemistry on a 492-01 HT model Procise protein sequencer (see
Fig. 1). Stock solutions of 100 mM
Tris(2-carboxyethyl)-phosphine hydrochloride (Pierce), and maleimide
(Aldrich) were prepared in 0.1 M ammonium acetate pH 4.5 and in 10% acetonitrile, respectively, and stored at 20 °C.
Peptide Synthesis
GID and analogues were manually assembled by Boc chemistry, deprotected, and cleaved from resin as described previously (21). Amino acid side-chain protection was as follows: Arg (Tos), Asn(Xan), Asp(OcHex), Cys(pMeBzl), Gla(OcHex), His (DNP), Hyp(Bzl), and Ser(Bzl). Linear peptides were manually assembled on t-Boc-cysteine-Pam resin, (0.75 mmol substitution/g; Applied Biosystems, Foster City, CA). Double couplings were used where necessary to achieve coupling efficiencies of >99.5%. HPLC-purified reduced peptides (50 µM) were oxidized in 100 mM ammonium bicarbonate at pH 7.5-8 with stirring for 24-48 h at 23 °C. The oxidized peptides were purified by preparative RP-HPLC. The major oxidized product was co-injected with native GID in a 2:1 ratio. Peptides were quantified initially in triplicate by amino acid analysis (22), then subsequently by RP-HPLC using an external reference standard for each peptide.
Functional Characterization of GID
RNA Preparation--
Plasmids containing cDNA encoding rat
3,
4,
7,
2, and
4 nAChR subunits were provided by J. Patrick (Baylor College of Medicine, Houston, TX) and subcloned into
the oocyte expression vector pNKS2 (23). cDNAs for the mouse
1,
1,
, and
subunits of the muscle nAChR were provided by Dr. V. Witzemann (Max-Planck Institute for Medical Research, Heidelberg,
Germany). Capped cRNAs were synthesized from linearized plasmid
cDNAs using the Message Machine Kit (Ambion, Austin, TX).
Expression in Xenopus Oocytes-- Oocytes were prepared as previously described (24), injected with 50 nl of cRNA (5-50 ng/µl), and kept at 19 °C in ND96 (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM Hepes at pH 7.4) supplemented with 50 mg/liter of gentamycin (Sigma Chemical Co.).
Two Electrode Voltage-Clamp Recording--
Two electrode
voltage-clamp recordings were performed in oocytes 2-10 days after
cRNA injection at a holding potential of 70 mV. Pipettes were pulled
from borosilicate glass (Harvard Apparatus Ltd., Kent, England) and
filled with 3 M KCl. Resistances were below 1 M
. Membrane currents were recorded using a two electrode virtual ground circuit on a GeneClamp 500B amplifier (Axon Instruments Inc., Union City, CA), filtered at 200 Hz and digitized at 1 kHz using
a Digidata 1322A interface and v8.2 Clampex software (Axon Instruments
Inc.). Recordings were performed in ND96 at room temperature. 1 µM ACh (Sigma) was used to activate
4
4 and
1
1
nAChRs, 100 µM ACh was used to activate
3
2,
3
4, and
4
2 nAChRs, and 100 µM
nicotine (Sigma) was used to activate
7 nAChR. The perfusion medium
was manually switched between ND96 with or without agonist using a
Valve Driver II (General Valve Corporation, Fairfield, NJ). A fast and
reproducible solution exchange (<300 ms) for agonist application was
achieved using a 50-µl funnel-shaped oocyte chamber combined with a
fast solution flow (~150 µl/s) fed through a custom-made manifold
mounted immediately above the oocyte. ACh pulses were applied for
2 s at 6-min intervals. After each application, the cell was
superfused for 1 min with agonist-free solution, and the flow was then
stopped for 5 min before agonist solution was re-introduced. Peptide
was applied when responses to three consecutive agonist applications
differed by less than 10%. After the 1-min agonist washout step was
stopped, 5.5 µl of a 10-fold concentrated peptide solution were
pipetted directly into the static bath, mixed by repeated pipetting,
and incubated for 5 min prior to application of agonist. Addition of
toxin directly to the recording chamber conserved material and avoided
potential adhesion of the toxin to tubing surfaces. To obtain estimates
of potency, dose-response curves were fitted to the data by the
equation percent response = 100/{1 + ([toxin]/IC50)nH} using Prism software
(GraphPad v 3.0 for Macintosh, San Diego, CA). To obtain estimates of
toxin washout kinetics, agonist responses were measured at 2-min
intervals under constant superfusion. For toxin application, oocytes
were superfused for 1 min before a 1-min incubation with the toxin
followed by the next agonist application.
NMR Solution Structure of GID
Sample Preparation-- Samples for 1H NMR measurement contained ~2.5 mM synthetic GID in 90% H2O, 10% D2O or 100% D2O at pH 2.8.
NMR Spectroscopy-- Spectra were obtained on a Bruker DMX 750 spectrometer at 280 and 287 K. All spectra were acquired in phase-sensitive mode using TPPI (25). The homonuclear spectra recorded included double quantum filtered DQF-COSY (26), TOCSY (27) using a MLEV17 spin lock sequence (28) with an isotropic mixing time of 80 ms; ECOSY (29), and NOESY (30) with mixing times of 150 and 350 ms. In DQF-COSY and ECOSY experiments, the water resonance was suppressed by low power irradiation during the relaxation delay (1.5 s). For the TOCSY and NOESY experiments, water suppression was achieved using a modified WATERGATE sequence (31). Two-dimensional spectra were generally collected over 4096 data points in the f2 dimension and 512 increments in the f1 dimension over a spectral width corresponding to 12 ppm. For identification of slowly exchanging amides, a series of one-dimensional and TOCSY spectra were run immediately after dissolving the sample in D2O. All spectra were processed on a Silicon Graphics workstation using XWINNMR (Bruker). The f1 dimension was zero-filled to 2048 real data points with the f1 and f2 dimensions being multiplied by a sine-squared function shifted by 90° prior to Fourier transformation. Processed spectra were analyzed and assigned using the program XEASY (32). Spectra were assigned using the sequential assignment protocol (33). The process was facilitated, in part, using the automatic assignment program NOAH, which is part of the DYANA package (34).
Structure Calculations--
Cross-peaks in NOESY spectra
recorded in 90% H2O, 10% D2O with mixing
times of 350 and 150 ms were integrated and calibrated in XEASY, and
distance constraints were derived using DYANA. Backbone dihedral angle
restraints were derived from
3JHNH coupling constants measured
from line shape analysis of antiphase cross-peak splitting in the
DQF-COSY spectrum. Angles were restrained to
120° ± 30 for
3JHNH
> 8.5 Hz and to
60° ± 30 for 3JHNH
< 5 Hz.
Stereospecific assignments of
-methylene protons and
1 dihedral
angles were derived from 3J
coupling
constants, measured from ECOSY spectra, in combination with NOE peak
intensities (35). Slowly exchanging amide protons identified by
D2O exchange experiments were used in conjunction with
preliminary structures to determine hydrogen bonds. In cases where
hydrogen bonds could be determined and unambiguously assigned, appropriate distance restraints were included in the subsequent calculations. Preliminary structures were calculated using a torsion angle simulated annealing protocol within DYANA. Final structures were
calculated using simulated annealing and energy minimization protocols
within CNS version 1.0 (36). The starting structures were generated
using random
,
dihedral angles and energy-minimized to produce
structures with the correct local geometry. A set of 50 structures was
generated by a torsion angle simulated annealing protocol (37, 38).
This protocol involves a high temperature phase comprising 4000 steps
of 0.015 ps of torsion angle dynamics, a cooling phase with 4000 steps
of 0.015 ps of torsion angle dynamics during which the temperature was
lowered to 0 K, and finally an energy minimization phase comprising
5000 steps of Powell minimization. Structures consistent with
restraints were subjected to further molecular dynamics and energy
minimization in a water shell, as described by Linge and Nilges (39).
The refinement in explicit water involves the following steps. First
heating to 500 K via steps of 100 K, each comprising 50 steps of 0.005 ps of Cartesian dynamics. Second, 2500 steps of 0.005 ps of Cartesian
dynamics at 500 K before a cooling phase where the temperature was
lowered in steps of 100 K, each comprising 2500 steps of 0.005 ps of
Cartesian dynamics. Finally, the structures were minimized with 2000 steps of Powell minimization. Structures were analyzed using Promotif and Procheck (40, 41).
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RESULTS |
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Isolation and Chemical Characterization of GID
Isolation and Sequence of GID--
In the search for new
-conotoxins, the crude venom of C. geographus was
screened for inhibition of agonist-evoked currents at muscle nAChR and
different combinations of neuronal nAChR subunits (
3
2,
3
4,
4
2,
4
4, and
7) heterologously expressed in
Xenopus oocytes. Crude venom (50 µg protein/ml by BCA
assay) caused 100% block of the
3
2 and
7 nAChR subtypes and
had weak activity at
4
2. LC/MS analysis of the crude venom, and
MS analysis of individual fractions, confirmed the presence of the
known muscle selective
-conotoxins GI, GIA, and GIB, while GII and
the neuronally active GIC were not found (Fig.
2, A and B). Thus
this pool of C. geographus crude venom appeared to contain a
novel neuronally active
-conotoxin. Inhibitory activity at the
3
2 and
7 subtypes was used to guide fractionations of the
crude venom, yielding a single component of 2184.9 Da (Fig. 2,
A and B) that inhibited
3
2,
7, and
4
2 nAChR subtypes. Edman sequencing of the reduced and alkylated
peptide, together with mass spectrometry evidence for the
-carboxyglutamic acid (
), revealed a new
4/7-conotoxin sequence IRD
CCSNPACRVNNOHVC (Fig. 1), that we named
-GID
following the nomenclature proposed by Olivera and co-workers (8). The identical sequence was also identified in Conus
tulipa crude venom.2 GID
possesses an NN(P/O) motif also found in
-conotoxins EpI, PnIA, and
PnIB (Fig. 1A) but additionally has a four residue
N-terminal tail. GID is the first non-amidated, neuronally active
-conotoxin identified.
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Chemical Synthesis of GID and Analogues--
To characterize GID
and investigate the influence of the N-terminal tail on its functional
properties, GID and an analogue missing the four N-terminal amino acids
(GID1-4) were synthesized (Fig. 1B). A single point
mutant, [R12A]GID, was also synthesized to investigate the influence
of the unusual charged residue in this position on nAChR selectivity.
All peptides were purified to >95% purity determined by HPLC and MS
analysis. Oxidized synthetic GID co-eluted with native GID on
reversed-phase HPLC (Fig. 2C), and both peptides had
identical masses (monoisotopic masses: observed native, 2184.9;
observed synthetic, 2184.9; theoretical, 2184.9).
Functional Characterization of GID--
Both native and synthetic
GID blocked 3
2 and
7 nAChRs receptors expressed in
Xenopus oocytes with equal potency (data not shown).
Synthetic GID was used in further experiments to determine the potency
and subtype selectivity of GID at neuronal
3
2,
3
4,
4
2,
4
4, and
7, and muscle
1
2
nAChRs
expressed in oocytes. The concentration response curves for GID are
shown in Fig. 4A, and IC50 values and Hill slope
coefficients are summarized in Table
I. GID was most potent at
3
2 and
7 nAChR subtypes, with IC50 values of 3 and 5 nM, respectively (Fig.
3A). The activity at the
3
4,
4
4, and muscle nAChRs was at least 1000-fold less. This
potency and selectivity profile of GID is somewhat reminiscent of
-conotoxin PnIA and [A10L]PnIA (42, 43). In contrast to these
peptides, however, GID also showed activity at the
4
2 subtype
(IC50 150 nM). Despite several sequence
similarities in the second loop, no
4
2 activity was found for
[A10L]PnIA (42) at 3 µM (Fig. 3B).
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Influence of the N-terminal Tail and Arg12 on Subtype
Selectivity--
To identify which structural determinants contribute
to GID selectivity, the roles of the N-terminal tail and the basic
residue Arg12 were investigated. Deletion of the four
N-terminal amino acids of GID reduced the activity of the peptide at
the 4
2 combination by ~4-fold (Fig. 3B).
Interestingly, GID
1-4 (1-10 µM) caused only a
partial block (~40%) of
4
2 receptors, indicating that the tail
of GID was required for full antagonist activity at this subtype
combination (no partial agonist activity was detected with this
truncated analogue, data not shown). However, potency at the
7 and
3
2 subtypes was little influenced by removal of the four
N-terminal residues. Activity of [R12A]GID at the
4
2 and
7
subtype was reduced ~10-fold compared with GID, whereas activity at
the
3
2 subtype was little affected (Fig. 3, B-D). An
analogue in which the four N-terminal amino acid residues of [R12A]GID were removed ([R12A]GID
1-4) did not cause any
inhibition of the
4
2 receptor at 3 µM (data not shown).
Influence of the N-terminal Sequence on the Off-rate
Kinetics--
Inhibition of the 4
2 nAChR by GID and its
analogues was rapidly reversible, with at least 90% recovery of
responses seen after 2 min (data not shown). However, recovery of the
3
2 and
7 subtypes from GID block was significantly slower,
requiring 10 and 15 min, respectively (Fig.
4, A and C). In
contrast, inhibition of both
3
2 and
7 receptors by GID
1-4
was reversed after a <2-min washout (Fig. 4, B and
D). Recovery from block by [A10L]PnIA, which does not have
an N-terminal tail, was rapid at the
3
2 subtype but slow at the
7 subtype (data not shown).
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Structural Characterization of GID
NMR Assignments-- All spectral data on GID were recorded in either 90% H2O, 10% D2O or 100% D2O. Spectra recorded at 287 K were primarily used for assignments, while spectra measured at other temperatures were used to resolve ambiguities.
Structure Determination and Analysis--
Analysis of the 350-ms
NOESY spectrum (750 MHz, 287 K) using the program XEASY allowed the
assignment of each spin system to a specific amino acid. All
non-intraresidual peaks were subsequently assigned both manually and
using the NOAH automatic assignment within the DYANA program package.
Interproton distance restraints were derived from the NOE intensities
and used in structure calculations using a torsion angle simulated
annealing protocol within DYANA. Preliminary structures were analyzed
to resolve spectral ambiguities and to facilitate the introduction of
new restraints. A set of restraints consisting of 183 NOE-derived
distances and 15 dihedral angle restraints was used in the final
calculations. These restraints included 82 sequential, 35 medium range,
10 long range, and 56 intraresidue distances, 6 angle restraints
(Cys5, Cys6, Arg12,
Val3, His17, and Cys19), and 9
1 angle restraints (Cys5,
Cys6, Asn8, Cys11,
Val13, Asn14, His17,
Val18, and Cys19). Side-chain angle restraints
were derived on the basis of coupling constants and NOE intensities
from a 150 ms NOESY spectrum. There were also four restraints included
for two hydrogen bonds identified in preliminary structures. In the
final round of structure calculations, these restraints were used to
calculate a family of 50 structures, using a torsion angle simulated
annealing (37, 38) protocol within CNS version 1.0 (36). Structures
consistent with the restraints were subjected to further molecular
dynamics and energy minimization in a water shell.
Secondary Structure--
The differences between the H chemical
shifts of GID and random coil values (44) are shown in Fig.
5. A negative secondary shift (upfield)
for several residues indicates that GID contains helical structural
elements. The longest uninterrupted region of negative secondary shifts
is observed for residues 9-13, suggesting that this region forms a
well-defined helix.
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Disulfide Connectivity--
The amino acid sequence of GID
includes four cysteine residues that are involved in two disulfide
bonds. Initially, the connectivities of the disulfide bonds were
unknown but were assumed to be identical to PnIA on the basis of
sequence similarities. Thus, the pairing of the disulfides was assumed
to be 5-11 and 6-19 (the globular conformation). To test the validity
of this assumption, in addition to calculations with the globular
connectivity, structures were calculated with the other two possible
disulfide connectivities, 5-6, 11-19 (beads conformation), and 5-19,
6-11 (ribbon conformation). The resultant structures for the beads and
ribbon isomers clearly violated the experimental data with the average
target function from DYANA significantly higher than observed for the
globular conformation. The beads and ribbon conformations had target
functions of 17.45 and 13.44, respectively, compared with 0.16 for the
globular conformation. Structures were also calculated without S-S
restraints, and the average target function was 6.94. Analysis of the
S-S distances in these structures revealed
Cys5-Cys11 was the most likely connectivity.
The next most likely bond was between
Cys6-Cys19; however, the distance between
Cys5-Cys19 was not significantly different. In
summary, the structure calculations with and without the disulfide
bonds confirmed that GID adopted the globular conformation, and thus
had the same disulfide bond pairings as other -conotoxins.
Description of the Three Dimensional Structure of GID-- A family of the 20 lowest NOE energy structures was chosen from the final set of 50 structures to represent the solution structure of GID. The statistics for these 20 structures, which had no distance violations greater than 0.2 Å and no dihedral angle violations greater than 3.0°, are given in Table II. The structures were well defined with the exception of the N-terminal tail, which had few experimental restraints defining this region. The mean RMSD over residues 4-19 was 0.34 ± 0.17 Å for the backbone atoms and 1.43 ± 0.32 Å for the heavy atoms. The family of structures obtained superimposed over the backbone atoms of residues 4-19 is shown in Fig. 6A. The ribbon representation of the lowest energy structure is shown in Fig. 6B. Analysis of the backbone angles reveals that 85% of the residues lie in the most favorable regions of the Ramachandran plot, 14% are in the additionally allowed region, 0.5% in the generously allowed region, and 0.5% in the disallowed region. The residues in the generously allowed and the disallowed regions correspond to Asp3 and Ile1, respectively, both in the poorly defined N-terminal tail.
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The solution structure of GID was determined to be a highly compact
globular structure consisting of a central region of -helix and
-turns at both the N and C termini. The
-helix comprises residues
9-13 and has two turns. H-bonds between
Asn8(O)-Cys11(HN) and
Pro9(O)-Val13(HN) were deduced from analysis
of preliminary structures and slow exchange data and were explicitly
included in the structure calculations. At the N terminus, the poor
definition of residues 1-3 may be associated with flexibility of this
region in solution. Analysis with PROMOTIF reveals that residues 5-8
are involved in a type I
-turn, while residues 15-18 are involved
in a type II
-turn in 7 of 20 final structures.
A surface representation of GID illustrates a distinct
hydrophobic face (Fig. 7A)
comprising residues Pro9, Ala10,
Val13, Hyp16, and Val18. The
hydrophilic residues on the surface include Ser7,
Asn8, Asn14, and Asn15. There are
also four charged residues on the surface of the molecule. Three
(Arg2, Asp3, and Gla4) are part of
the disordered N-terminal tail. The fourth (Arg12) is
situated adjacent to the hydrophobic face.
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DISCUSSION |
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Using functional nAChR assay-guided fractionation, we isolated
-conotoxin GID from the crude venom of C. geographus.
-GID is the sixth
-conotoxin and the second neuronally active
-conotoxin isolated from this species (8, 45), showing the
importance of
-conotoxins targeting both muscle and neuronal nAChRs
for prey capture. GID appears to replace GIC in this pool of C. geographus venom. The sequence of GID differs from previously
described
-conotoxins in a number of regards. The most striking is
the novel N-terminal tail comprising four amino acids
(Ile1, Arg2, Asp3,
Gla4) including the post-translationally modified
-carboxyglutamic acid (Gla). An examination of the pre-pro regions
of
-conotoxins from the SWISS-PROT data base reveals that most
conopeptide precursor sequences contain Arg-Arg, Lys-Lys, Gln-Arg,
Lys-Arg, or Arg-Asp motifs as the cleavage site. For example, ImIIA
utilizes Ile-Arg-Asp-Tyr (accession number Q9U619) to leave a Tyr at
position 1 in the mature conopeptide. Since the Arg-Asp cleavage site
is not utilized in GID, it appears that the
-carboxylation of Glu4
in GID inhibits this cleavage site, allowing the N-terminal tail to
survive in the venom. GID is the first
-conotoxin to utilize a
hydroxyproline residue, despite its widespread use in other classes of
conotoxins, and is the first neuronally active
-conotoxin with a
non-amidated C terminus.
Several neuronally active -conotoxins have been identified that have
nanomolar potency for only the
3
2 (MII, GIC),
3
4 (AuIB), or
7 (ImI) receptors expressed in Xenopus oocytes (10, 45,
46, 47) (Fig. 1). Other
-conotoxins such as PnIA and PnIB inhibit
both the
7 and
3
2 subtypes with nanomolar potencies, with PnIA
being
3
2 selective and PnIB being
7 selective (42, 43). GID
also has low nanomolar potency at these two nAChR subtypes, but
additionally targets the
4
2 receptor at nanomolar levels. Thus
GID has structural features that allow it to recognize a broader range
of nAChR binding sites than other
-conotoxins, making it a versatile
tool for structure-activity studies at the ACh binding site. Comparison
of its structure with those of more "specialized"
-conotoxins
may reveal the critical determinants for subtype selectivity.
The ACh binding site has been localized at the interface of an
-subunit (+ face) and the respective non-
subunit (or
face in case of the
7 nAChRs) (48-50). To date, there is limited
information on the binding mode of neuronally active
-conotoxins and
the factors that determine subtype selectivity. In a study to determine the direct interactions between amino acids of the
7 receptor and
-conotoxin ImI, Quiram et al. (49) found that the two
exposed loops of the peptide interact with different subunits of the
7 receptor, thus bridging the interface between two adjacent
subunits. However, a similar mode of binding was not found for PnIB
(51). Instead, the key bioactive residues of this peptide form a
localized hydrophobic patch that appears to interact mostly with the + face of the
7 binding site. Various neuronally active
-conotoxins might have evolved that have one or more attachment points and target
different microdomains that overlap the ACh binding site on nAChRs
(51). Therefore, it might be useful to further subgroup the neuronally
active
-conotoxins based on their subunit specificity and sequence
similarity in order to compare structures that are likely to have
similar binding modes.
The three-dimensional solution structure of -conotoxin GID reveals
that it is a well defined molecule, with a backbone RMSD over residues
4-19 of 0.34 Å; however, residues 1-3 are completely disordered. The
side-chains are well defined, with the exception of Arg2,
Gla4, and Arg12. The core of the molecule is
occupied by the cysteine residue side-chains, whereas the side-chains
of the remaining residues are solvent-exposed. The NMR structure of GID
was compared with the crystal structure of PnIA (17), an
-conotoxin
with the same global fold and related activity at
7 and
3
2
(Fig. 7B). Superimposition of the
-helical backbone
residues of GID with PnIA gives an RMS deviation of 0.29 Å. This
similarity is evidence that
4/7-conotoxins utilize the same highly
conserved, well-defined backbone structure to present a range of
different side-chains for interaction with specific nAChRs.
Charge distribution appears to be an important factor that determines
nAChR selectivity, with neuronal specific -conotoxins being neutral
or negatively charged, and muscle-specific
-conotoxins having a net
positive charge (52). For example, PnIA contains a single negatively
charged residue, Asp14, which together with a positive N
terminus and neutral, amidated C terminus results in a net charge of
zero. At physiological pH, GID has four charged residues including two
positive (Arg2 and Arg12) and two negatively
charged residues (Asp3 and Gla4). Combined with
balancing positive and negatively charged termini (unlike most other
conotoxins GID contains no amidated C terminus) the net charge is close
to zero. A comparison of the surfaces of GID and PnIA shows that both
molecules have a similar hydrophobic patch on one face of the molecule.
This patch includes amino acid residues Pro6,
Pro7, Ala10, and Pro13 in PnIA
(Fig. 7B), and the corresponding residues Pro9,
Ala10, Val13, and O16 in GID (Fig.
7A). A similar cluster of hydrophobic residues
(Leu5, Pro6, Pro7,
Ala9, and Leu10) has been shown to be important
for the high affinity binding of PnIB to the
7 receptor (51).
Exchanging Ala with Leu at position 10 in PnIA ([A10L]PnIA) enhanced
7 and reduced
3
2 potency, suggesting that a long aliphatic
side-chain in this position favored
7 selectivity (42, 43). In
support, the
4/7-conotoxin GIC has a Gly in position 10 and is a
potent
3
2 inhibitor (45) (see Table
III). GID, which has similar potency at
both
7 and
3
2 receptors, has an intermediate length side-chain
(Val13) in the equivalent position to Ala and Leu. Taken
together, it appears that the length of the side-chain in position 10 (or position 13 in GID) of these
4/7-conotoxins correlates with
their selectivity for
7 or
3
2 receptors: the longer the
aliphatic side-chain the more
7-selective and less
3
2-selective the peptides become (Table III).
|
The neighboring amino acid residues in positions 9 and 11 also seem to
contribute to this selectivity: Arg12 of GID (equivalent to
Ala9 of PnIA), contributes to 7 selectivity, since
replacing it with an Ala reduced
7 activity by 10-fold without
affecting activity at the
3
2 receptor. In contrast,
Asn11 may contribute more to
3
2 versus
7 selectivity, since [N11S]PnIA was 24-fold less active at the
3
2 combination but only 7-fold less active at the
7 subtype as
compared with PnIA. Likewise, PnIB, which has a Ser in position 11, was
~20-fold less active at
3
2 receptors but only 5-fold less
active at the
7 nAChR than [A10L]PnIA (43). Activity at
7 was
little altered in the Asn12Ala and Pro13Hyp analogues of PnIB (51).
However, their conservation in most
4/7-conotoxins suggests they
have evolved as a structurally important motif. As positions 14 and 15 in PnIB also had little influence on affinity (51), it appears that the
side-chains of the first three N-terminal residues in loop II of these
4/7-conotoxins play a key role in determining potency and
selectivity at
3
2 and
7 subtypes.
GID contains a unique N-terminal tail comprising four amino acids.
Removal of these residues did not affect activity at 7 and
3
2
subtypes but strongly reduced block at the
4
2 subtype, turning
GID into a partial inhibitor at this receptor. These results suggest
that N-terminal residues outside the cysteine framework can contribute
to
4
2 activity. However, this N-terminal motif is not essential
for full inhibition of
4
2, since GIC is a full inhibitor of the
4
2 receptor with an IC50 value of 300 nM
(45). The reduced times to washout of GID
1-4 from the
7 and
3
2 receptors suggest that the N-terminal tail can stabilize the
binding of GID to
7 and
3
2 receptors, presumably by
interacting with residues near the ACh binding pocket that are not
accessible to smaller
-conotoxins. The reduced activity of
[R12A]GID at the
4
2 receptor indicates that Arg12
in loop II also contributes to GID block of
4
2. Thus GID reveals novel features contributing to
-conotoxin binding to the
4
2 receptor. Improving the structural stability of the N-terminal tail in
GID or adding it to other
-conotoxins provides new avenues for the
development of
4
2 selective inhibitors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Trudy Bond for amino acid analysis, Alun Jones for LC/MS analysis, Roger Pearson for peptide sequencing, and Richard Clark for critical reading of the article.
![]() |
FOOTNOTES |
---|
* This study was supported by Australian Research Council Discovery Grant DP0208295 and by an Australian Research Council Special Research Center for Functional and Applied Genomics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1MTQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Supported by Research Fellowship NI 592/2-1 of the Deutsche Forschungsgemeinschaft.
** To whom correspondence should be addressed: Inst. for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. Tel.: 61-7-3365-1924; Fax: 61-7-3365-1990; E-mail: r.lewis@imb.uq.edu.au.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M210280200
2 L. Thomas and R. J. Lewis, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
nAChR, nicotinic
acetylcholine receptors;
ACh, acetylcholine;
-GID,
-conotoxin
GID;
-PnIA,
-conotoxin PnIA;
,
-carboxyglutamic acid (Gla);
O, hydroxyproline (Hyp);
RMSD, root mean-squared deviation;
RP-HPLC, reverse phase-high performance liquid chromatography.
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