Isolation, Structure, and Activity of GID, a Novel alpha 4/7-Conotoxin with an Extended N-terminal Sequence*

Annette NickeDagger §, Marion L. LoughnanDagger , Emma L. MillardDagger §, Paul F. AlewoodDagger , David J. Adams§, Norelle L. DalyDagger , David J. CraikDagger ||, and Richard J. LewisDagger §**

From the Dagger  Institute for Molecular Bioscience and § School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

Received for publication, October 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha -conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha -conotoxins, alpha -GID has a four amino acid N-terminal tail, gamma -carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3beta 2 nAChRs with IC50 values of 5 and 3 nM, respectively and is at least 1000-fold less potent at the alpha 1beta 1gamma delta , alpha 3beta 4, and alpha 4beta 4 combinations. GID also potently inhibits the alpha 4beta 2 subtype (IC50 of 150 nM). Deletion of the N-terminal sequence (GIDDelta 1-4) significantly decreased activity at the alpha 4beta 2 nAChR but hardly affected potency at alpha 3beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg12 contributed to alpha 4beta 2 and alpha 7 activity but not to alpha 3beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha -conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 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 alpha 7 versus alpha 3beta 2 selectivity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  and three beta  subunits (alpha 2-alpha 7, alpha 9, alpha 10, beta 2-beta 4) of the nAChRs have been cloned from sensory and neuronal mammalian cells (2-4). For the alpha 7 and alpha 9 subunits, it has been shown that they need no additional subunits to form functional ion channels upon heterologous expression. All other alpha  subunits, however, require at least the co-expression of one beta  subunit, or another alpha  subunit in the case of alpha 10. Ternary combinations of two different alpha  and one beta  subunit or two different beta  and one alpha  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 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. alpha -Conotoxins are competitive antagonists of acetylcholine (ACh) binding to the nAChR (7). The alpha -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 alpha 3/5, alpha 4/7, alpha 4/6, and alpha 4/3 structural subfamilies (8). The alpha 3/5-conotoxins are selective for the muscle-type nAChR, while most alpha 4/7- and alpha 4/6-conotoxins are selective for neuronal nAChRs. An exception is the alpha 4/7-conotoxin EI, which binds to the alpha /delta interface of the muscle-type nAChR (9). The alpha 4/3-type characterized by ImI is alpha 7-selective (10). The three-dimensional structures of different neuronal-specific and muscle-specific alpha -conotoxins have been determined by NMR (11-15) and x-ray structural analysis (16-19). The alpha 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.


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Fig. 1.   Comparison of alpha -GID and related neuronally active alpha -conotoxins. A, sequence and selectivity of neuronally active alpha -conotoxins. A motif (NN(O/P)) commonly found in the second loop of alpha 4/7-conotoxins is marked in gray. Asterisks indicate C-terminal amidation. Sulfated Tyr are underlined, gamma , gamma -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 alpha 4beta 2 nAChRs together with the alpha 7 subtype are the most abundant nAChRs in mammalian brain. Knockout studies in mice have revealed an important role for alpha 4 and beta 2 in pain and cognition (20) but further investigations of its role are limited by the lack of alpha 4beta 2-selective inhibitors. A primary goal of this study was to identify new alpha -conotoxins active at the alpha 4beta 2 subtype. In this report, we describe the isolation and characterization of alpha 4/7-conotoxin GID. GID possesses a novel four amino acid N-terminal tail and an Arg in position 12 that contribute to alpha 4beta 2 selectivity. The NMR structure of GID was determined to define the location of structural features that contribute to the selectivity of GID.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 3, alpha 4, alpha 7, beta 2, and beta 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 alpha 1, beta 1, gamma , and delta  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 MOmega . 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 alpha 4beta 4 and alpha 1beta 1gamma delta nAChRs, 100 µM ACh was used to activate alpha 3beta 2, alpha 3beta 4, and alpha 4beta 2 nAChRs, and 100 µM nicotine (Sigma) was used to activate alpha 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 3JHNHalpha coupling constants measured from line shape analysis of antiphase cross-peak splitting in the DQF-COSY spectrum. Angles were restrained to -120° ± 30 for 3JHNHalpha  > 8.5 Hz and to -60° ± 30 for 3JHNHalpha  < 5 Hz. Stereospecific assignments of beta -methylene protons and chi 1 dihedral angles were derived from 3Jalpha beta 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 phi , psi  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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation and Chemical Characterization of GID

Isolation and Sequence of GID-- In the search for new alpha -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 (alpha 3beta 2, alpha 3beta 4, alpha 4beta 2, alpha 4beta 4, and alpha 7) heterologously expressed in Xenopus oocytes. Crude venom (50 µg protein/ml by BCA assay) caused 100% block of the alpha 3beta 2 and alpha 7 nAChR subtypes and had weak activity at alpha 4beta 2. LC/MS analysis of the crude venom, and MS analysis of individual fractions, confirmed the presence of the known muscle selective alpha -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 alpha -conotoxin. Inhibitory activity at the alpha 3beta 2 and alpha 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 alpha 3beta 2, alpha 7, and alpha 4beta 2 nAChR subtypes. Edman sequencing of the reduced and alkylated peptide, together with mass spectrometry evidence for the gamma -carboxyglutamic acid (gamma ), revealed a new alpha 4/7-conotoxin sequence IRDgamma CCSNPACRVNNOHVC (Fig. 1), that we named alpha -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 alpha -conotoxins EpI, PnIA, and PnIB (Fig. 1A) but additionally has a four residue N-terminal tail. GID is the first non-amidated, neuronally active alpha -conotoxin identified.


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Fig. 2.   Isolation and synthesis of alpha -GID. A, total ion chromatogram of C. geographus crude venom extract. The arrow indicates alpha -GID. A reconstructed molecular mass spectrum of GID is shown in the inset. B, purification of GID by HPLC. C, coelution of native and synthetic GID. Synthetic GID (sGID) and native (nGID) were co-injected in a 2:1 ratio and analyzed by HPLC. Retention time (38.3 min) and peak width was the same for synthetic, native, and the co-injected peptides.

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 (GIDDelta 1-4) 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 alpha 3beta 2 and alpha 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 alpha 3beta 2, alpha 3beta 4, alpha 4beta 2, alpha 4beta 4, and alpha 7, and muscle alpha 1beta 2gamma delta 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 alpha 3beta 2 and alpha 7 nAChR subtypes, with IC50 values of 3 and 5 nM, respectively (Fig. 3A). The activity at the alpha 3beta 4, alpha 4beta 4, and muscle nAChRs was at least 1000-fold less. This potency and selectivity profile of GID is somewhat reminiscent of alpha -conotoxin PnIA and [A10L]PnIA (42, 43). In contrast to these peptides, however, GID also showed activity at the alpha 4beta 2 subtype (IC50 150 nM). Despite several sequence similarities in the second loop, no alpha 4beta 2 activity was found for [A10L]PnIA (42) at 3 µM (Fig. 3B).

                              
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Table I
Inhibition of nAChR currents by alpha -GID and analogues
IC50 values (nM) and Hill slopes (-nH) for inhibition of agonist-evoked current through alpha 3beta 2, alpha 4beta 2, and alpha 7 subtypes expressed in Xenopus oocytes are shown.


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Fig. 3.   Comparison of subtype selectivity and activity of alpha -GID and its analogues at nAChRs expressed in Xenopus oocytes. Oocytes were injected with cRNA encoding the indicated nAChR subunits and voltage clamped 2-10 days after injection. Responses to ACh or nicotine (for alpha 7) after a 5-min incubation in the presence of peptide are shown as percentage of control responses. Error bars are S.E. and n = 3-5 for each data point. A, subtype selectivity of GID. Comparison of inhibition by GID and its analogues at alpha 4beta 2 (B), alpha 3beta 2 (C), and alpha 7 (D) nAChRs. IC50 values and Hill slope coefficients are summarized in Table I.

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 alpha 4beta 2 combination by ~4-fold (Fig. 3B). Interestingly, GIDDelta 1-4 (1-10 µM) caused only a partial block (~40%) of alpha 4beta 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 alpha 7 and alpha 3beta 2 subtypes was little influenced by removal of the four N-terminal residues. Activity of [R12A]GID at the alpha 4beta 2 and alpha 7 subtype was reduced ~10-fold compared with GID, whereas activity at the alpha 3beta 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]GIDDelta 1-4) did not cause any inhibition of the alpha 4beta 2 receptor at 3 µM (data not shown).

Influence of the N-terminal Sequence on the Off-rate Kinetics-- Inhibition of the alpha 4beta 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 alpha 3beta 2 and alpha 7 subtypes from GID block was significantly slower, requiring 10 and 15 min, respectively (Fig. 4, A and C). In contrast, inhibition of both alpha 3beta 2 and alpha 7 receptors by GIDDelta 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 alpha 3beta 2 subtype but slow at the alpha 7 subtype (data not shown).


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Fig. 4.   Washout kinetics of GID and GIDDelta 1-4 from alpha 3beta 2 and alpha 7 nAChRs. Xenopus oocytes expressing the alpha 3beta 2 (A and B) or alpha 7 (C and D) nAChR subtypes were stimulated with 1-s pulses of ACh or nicotine (in the case of alpha 7) applied at 2-min intervals. Oocytes were first perfused for one more minute with ND96 solution, then incubated with toxin for 1 min in a static bath, before recommencing agonist applications during superfusion. Experiments were repeated three times with similar results. Note that at short incubation times GID is less potent than GIDDelta 1-4, indicating that removal of the N-terminal tail increased on-rate kinetics as well as accelerating the off-rate kinetics.

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 phi  angle restraints (Cys5, Cys6, Arg12, Val3, His17, and Cys19), and 9 chi 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 Halpha 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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Fig. 5.   Halpha chemical shift differences of alpha -GID from random coil values.

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 alpha -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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Table II
Geometric and energy statistics for the 20 final structures of alpha -GID


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Fig. 6.   The three-dimensional structure of alpha -GID. A, a family of 20 GID structures superimposed over the backbone atoms of residues 4-19. The disulfides are shown in red and the N and C termini are labeled. B, secondary structure of GID showing the alpha -helix, two beta  turns between residues 4-7 and 14-17, and the tail region. The N and C termini are labeled. The coordinates for alpha -GID have been deposited with the Research Collaboratory for Structural Bioinformatics Protein Data bank (PDB 1MTQ).

The solution structure of GID was determined to be a highly compact globular structure consisting of a central region of alpha -helix and beta -turns at both the N and C termini. The alpha -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 beta -turn, while residues 15-18 are involved in a type II beta -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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Fig. 7.   Comparison of the surfaces of alpha -GID (A) and PnIA (B). Two orientations (rotated 180° around the y-axis) are shown for each molecule superimposed over the alpha -helical residues. Negatively and positively charged residues are shown in red and blue, respectively (the His is assumed to be uncharged at physiological pH). Hydrophobic residues are shown in green, and hydrophilic residues are in gray. Cysteine residues involved in disulfide bonds are shown in yellow. Note a common hydrophobic patch comprising Pro6, Pro7, Ala10, and Pro13 in PnIA, and Pro9, Ala10, Val13, and Hyp16 in GID.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using functional nAChR assay-guided fractionation, we isolated alpha -conotoxin GID from the crude venom of C. geographus. alpha -GID is the sixth alpha -conotoxin and the second neuronally active alpha -conotoxin isolated from this species (8, 45), showing the importance of alpha -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 alpha -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 gamma -carboxyglutamic acid (Gla). An examination of the pre-pro regions of alpha -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 gamma -carboxylation of Glu4 in GID inhibits this cleavage site, allowing the N-terminal tail to survive in the venom. GID is the first alpha -conotoxin to utilize a hydroxyproline residue, despite its widespread use in other classes of conotoxins, and is the first neuronally active alpha -conotoxin with a non-amidated C terminus.

Several neuronally active alpha -conotoxins have been identified that have nanomolar potency for only the alpha 3beta 2 (MII, GIC), alpha 3beta 4 (AuIB), or alpha 7 (ImI) receptors expressed in Xenopus oocytes (10, 45, 46, 47) (Fig. 1). Other alpha -conotoxins such as PnIA and PnIB inhibit both the alpha 7 and alpha 3beta 2 subtypes with nanomolar potencies, with PnIA being alpha 3beta 2 selective and PnIB being alpha 7 selective (42, 43). GID also has low nanomolar potency at these two nAChR subtypes, but additionally targets the alpha 4beta 2 receptor at nanomolar levels. Thus GID has structural features that allow it to recognize a broader range of nAChR binding sites than other alpha -conotoxins, making it a versatile tool for structure-activity studies at the ACh binding site. Comparison of its structure with those of more "specialized" alpha -conotoxins may reveal the critical determinants for subtype selectivity.

The ACh binding site has been localized at the interface of an alpha -subunit (+ face) and the respective non-alpha subunit (or - face in case of the alpha 7 nAChRs) (48-50). To date, there is limited information on the binding mode of neuronally active alpha -conotoxins and the factors that determine subtype selectivity. In a study to determine the direct interactions between amino acids of the alpha 7 receptor and alpha -conotoxin ImI, Quiram et al. (49) found that the two exposed loops of the peptide interact with different subunits of the alpha 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 alpha 7 binding site. Various neuronally active alpha -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 alpha -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 alpha -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 alpha -conotoxin with the same global fold and related activity at alpha 7 and alpha 3beta 2 (Fig. 7B). Superimposition of the alpha -helical backbone residues of GID with PnIA gives an RMS deviation of 0.29 Å. This similarity is evidence that alpha 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 alpha -conotoxins being neutral or negatively charged, and muscle-specific alpha -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 alpha 7 receptor (51). Exchanging Ala with Leu at position 10 in PnIA ([A10L]PnIA) enhanced alpha 7 and reduced alpha 3beta 2 potency, suggesting that a long aliphatic side-chain in this position favored alpha 7 selectivity (42, 43). In support, the alpha 4/7-conotoxin GIC has a Gly in position 10 and is a potent alpha 3beta 2 inhibitor (45) (see Table III). GID, which has similar potency at both alpha 7 and alpha 3beta 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 alpha 4/7-conotoxins correlates with their selectivity for alpha 7 or alpha 3beta 2 receptors: the longer the aliphatic side-chain the more alpha 7-selective and less alpha 3beta 2-selective the peptides become (Table III).

                              
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Table III
Comparison of loop II consensus sequences for selected alpha 4/7-conotoxins and analogues
IC50 values and selectivity at alpha 7 and alpha 3beta 2 subtypes expressed in Xenopus oocytes are reported.

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 alpha 7 selectivity, since replacing it with an Ala reduced alpha 7 activity by 10-fold without affecting activity at the alpha 3beta 2 receptor. In contrast, Asn11 may contribute more to alpha 3beta 2 versus alpha 7 selectivity, since [N11S]PnIA was 24-fold less active at the alpha 3beta 2 combination but only 7-fold less active at the alpha 7 subtype as compared with PnIA. Likewise, PnIB, which has a Ser in position 11, was ~20-fold less active at alpha 3beta 2 receptors but only 5-fold less active at the alpha 7 nAChR than [A10L]PnIA (43). Activity at alpha 7 was little altered in the Asn12Ala and Pro13Hyp analogues of PnIB (51). However, their conservation in most alpha 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 alpha 4/7-conotoxins play a key role in determining potency and selectivity at alpha 3beta 2 and alpha 7 subtypes.

GID contains a unique N-terminal tail comprising four amino acids. Removal of these residues did not affect activity at alpha 7 and alpha 3beta 2 subtypes but strongly reduced block at the alpha 4beta 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 alpha 4beta 2 activity. However, this N-terminal motif is not essential for full inhibition of alpha 4beta 2, since GIC is a full inhibitor of the alpha 4beta 2 receptor with an IC50 value of 300 nM (45). The reduced times to washout of GIDDelta 1-4 from the alpha 7 and alpha 3beta 2 receptors suggest that the N-terminal tail can stabilize the binding of GID to alpha 7 and alpha 3beta 2 receptors, presumably by interacting with residues near the ACh binding pocket that are not accessible to smaller alpha -conotoxins. The reduced activity of [R12A]GID at the alpha 4beta 2 receptor indicates that Arg12 in loop II also contributes to GID block of alpha 4beta 2. Thus GID reveals novel features contributing to alpha -conotoxin binding to the alpha 4beta 2 receptor. Improving the structural stability of the N-terminal tail in GID or adding it to other alpha -conotoxins provides new avenues for the development of alpha 4beta 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.

|| An ARC Professorial Fellow.

** 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; alpha -GID, alpha -conotoxin GID; alpha -PnIA, alpha -conotoxin PnIA; gamma , gamma -carboxyglutamic acid (Gla); O, hydroxyproline (Hyp); RMSD, root mean-squared deviation; RP-HPLC, reverse phase-high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lloyd, G. K., and Williams, M. (2000) J. Pharmacol. Exp. Ther. 292, 461-467[Free Full Text]
2. McGehee, D. S., and Role, L. W. (1995) Annu. Rev. Physiol. 57, 521-546[CrossRef][Medline] [Order article via Infotrieve]
3. Sargent, P. B. (1993) Annu. Rev. Neurosci. 16, 403-443[CrossRef][Medline] [Order article via Infotrieve]
4. Elgoyhen, A. B., Vetter, D. E., Katz, E., Rothlin, C. V., Heinemann, S. F., and Boulter, J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3501-3506[Abstract/Free Full Text]
5. Colquhoun, L. M., and Patrick, J. W. (1997) J. Neurochem. 69, 2356-2362
6. Sivilotti, L. G., McNeil, D. K., Lewis, T. M, Nassar, M. A., Schoepfer, R., and Colquhoun, D. (1997) J. Physiol. (Lond.) 500, 123-138[Abstract]
7. Dwoskin, L. P., and Crooks, P. A. (2001) J. Pharmcol. Exp. Ther. 298, 395-402[Abstract/Free Full Text]
8. McIntosh, J. M., Santos, A. D., and Olivera, B. M. (1999) Annu. Rev. Biochem. 68, 59-88[CrossRef][Medline] [Order article via Infotrieve]
9. Martinez, J. S., Olivera, B. M., Gray, W. R., Craig, A. G., Groebe, D. R., Abramson, S. N., and McIntosh, J. M. (1995) Biochemistry 34, 14519-14526[Medline] [Order article via Infotrieve]
10. Johnson, D. S., Martinez, J., Elgoyhen, A. B., Heinemann, S. F., and McIntosh, J. M. (1995) Mol. Pharmacol. 48, 194-199[Abstract]
11. Gehrmann, J., Alewood, P. F., and Craik, D. J. (1998) J. Mol. Biol. 278, 401-415[CrossRef][Medline] [Order article via Infotrieve]
12. Gehrmann, J., Daly, N. L., Alewood, P. F., and Craik, D. J. (1999) J. Med. Chem. 42, 2364-2372[CrossRef][Medline] [Order article via Infotrieve]
13. Hill, J. M., Oomen, C. J., Miranda, L. P., Bingham, J. P., Alewood, P. F., and Craik, D. J. (1998) Biochemistry 37, 15621-15630[CrossRef][Medline] [Order article via Infotrieve]
14. Rogers, J. P., Luginbuhl, P., Shen, G. S., McCabe, R. T., Stevens, R. C., and Wemmer, D. E. (1999) Biochemistry 38, 3874-3882[CrossRef][Medline] [Order article via Infotrieve]
15. Shon, K. J., Koerber, S. C., Rivier, J. E., Olivera, B. M., and McIntosh, J. M. (1997) Biochemistry 36, 15693-15700[CrossRef][Medline] [Order article via Infotrieve]
16. Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B., and Gray, W. R. (1996) Biochemistry 35, 11329-11335[CrossRef][Medline] [Order article via Infotrieve]
17. Hu, S. H., Gehrmann, J., Guddat, L. W., Alewood, P. F., Craik, D. J., and Martin, J. L. (1996) Structure 4, 417-423[Medline] [Order article via Infotrieve]
18. Hu, S. H., Gehrmann, J., Alewood, P. F., Craik, D. J., and Martin, J. L. (1997) Biochemistry 36, 11323-11330[CrossRef][Medline] [Order article via Infotrieve]
19. Hu, S. H., Loughnan, M., Miller, R., Weeks, C. M., Blessing, R. H., Alewood, P. F., Lewis, R. J., and Martin, J. L. (1998) Biochemistry 37, 11425-11433[CrossRef][Medline] [Order article via Infotrieve]
20. Cordero-Erausquin, M., Marubio, L. M., Klink, R., and Changeux, J. P. (2000) Trends Pharmacol. Sci. 21, 211-217[CrossRef][Medline] [Order article via Infotrieve]
21. Schnölzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S. B. (1992) Int. J. Pept. Protein Res. 40, 180-193[Medline] [Order article via Infotrieve]
22. Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) J. Chromatogr. 336, 93-104[Medline] [Order article via Infotrieve]
23. Gloor, S., Pongs, O., and Schmalzing, G. (1995) Gene (Amst.) 160, 213-217[CrossRef][Medline] [Order article via Infotrieve]
24. Nicke, A., Bäumert, H. G., Rettinger, J., Eichele, A., Lambrecht, G., Mutschler, E., and Schmalzing, G. (1998) EMBO J. 17, 3016-3028[Abstract/Free Full Text]
25. Marion, D., and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 967-974[Medline] [Order article via Infotrieve]
26. Rance, M., Sorensen, O. W., Bodenhausen, G., Wagner, G., Ernst, R. R., and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun. 117, 479-485[Medline] [Order article via Infotrieve]
27. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528
28. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 56, 355-360
29. Griesinger, C., Sorensen, O. W., and Ernst, R. R. (1987) J. Magn. Reson. 75, 474-492
30. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553[CrossRef]
31. Piotto, M., Saudek, V., and Sklenar, V. (1992) J. Biomol. NMR 2, 661-665[Medline] [Order article via Infotrieve]
32. Eccles, C., Guntert, P., Billeter, M., and Wüthrich, K. (1991) J. Biomol. NMR 1, 111-130[Medline] [Order article via Infotrieve]
33. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids , Wiley-Interscience, New York
34. Guntert, P., Mumenthaler, C., and Wüthrich, K. (1997) J. Mol. Biol. 273, 283-298[CrossRef][Medline] [Order article via Infotrieve]
35. Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 51-55[CrossRef][Medline] [Order article via Infotrieve]:29-32
36. Brünger, A. T., Adams, P. D., and Rice, L. M. (1997) Structure 5, 325-336[Medline] [Order article via Infotrieve]
37. Rice, L. M., and Brünger, A. T. (1994) Proteins 19, 277-290[Medline] [Order article via Infotrieve]
38. Stein, E. G., Rice, L. M., and Brünger, A. T. (1997) J. Magn. Reson. 124, 154-164[CrossRef][Medline] [Order article via Infotrieve]
39. Linge, J. P., and Nilges, M. (1999) J. Biomol. NMR 13, 51-59[CrossRef][Medline] [Order article via Infotrieve]
40. Hutchinson, E. G., and Thornton, J. M. (1996) Protein Sci. 5, 212-220[Abstract/Free Full Text]
41. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, 477-486[Medline] [Order article via Infotrieve]
42. Hogg, R. C., Miranda, L. P., Craik, D. J., Lewis, R. J., Alewood, P. F., and Adams, D. J. (1999) J. Biol. Chem. 274, 36559-36564[Abstract/Free Full Text]
43. Luo, S., Nguyen, T. A., Cartier, G. E., Olivera, B. M., Yoshikami, D., and McIntosh, J. M. (1999) Biochemistry 38, 14542-14548[CrossRef][Medline] [Order article via Infotrieve]
44. Wishart, D. S., Bigam, C. G., Holm, A., Hodges, R. S., and Sykes, B. D. (1995) J. Biomol. NMR 5, 67-81[Medline] [Order article via Infotrieve]
45. McIntosh, J. M., Dowell, C., Watkins, M., Garret, J. E., Yoshikami, D., and Olivera, B. M. (2002) J. Biol. Chem. 277, 33610-33615[Abstract/Free Full Text]
46. Cartier, G. E., Yoshikami, D., Gray, W. R., Luo, S., Olivera, B. M., and McIntosh, J. M. (1996) J. Biol. Chem. 271, 7522-7528[Abstract/Free Full Text]
47. Luo, S., Kulak, J. M., Cartier, G. E., Jacobsen, R. B., Yoshikami, D., Olivera, B. M., and McIntosh, J. M. (1998) J. Neurosci. 18, 8571-8579[Abstract/Free Full Text]
48. Hucho, F., Tsetlin, V. I., and Machold, J. (1996) Eur. J. Biochem. 239, 539-557[Abstract]
49. Quiram, P. A., Jones, J. J., and Sine, S. M. (1999) J. Biol. Chem. 274, 19517-19524[Abstract/Free Full Text]
50. Karlin, A. (2002) Nat. Rev. Neurosci. 3, 102-114[CrossRef][Medline] [Order article via Infotrieve]
51. Quiram, P. A., McIntosh, J. M., and Sine, S. M. (2000) J. Biol. Chem. 275, 4889-4896[Abstract/Free Full Text]
52. Rogers, J. P., Luginbuhl, P., Pemberton, K., Harty, P., Wemmer, D. E., and Stevens, R. C. (2000) J. Mol. Biol. 304, 911-926[CrossRef][Medline] [Order article via Infotrieve]
53. Loughnan, M., Bond, T., Atkins, A., Cuevas, J., Adams, D. J., Broxton, N., Livett, B., Down, J., Jones, A., Alewood, P. F., and Lewis, R. J. (1998) J. Biol. Chem. 273, 15667-15674[Abstract/Free Full Text]


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