From the Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The neuronal-specific toxin -conotoxin ImI
(CTx ImI) has the sequence
Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a
constrained two-loop structure. To investigate the structural basis for
bioactivity we mutated individual residues in CTx ImI and determined
bioactivity. Bioactivity of the toxins was determined by their
competition against 125I-labeled
-bungarotoxin binding
to homomeric receptors containing
7 sequence in the
major extracellular domain and 5HT-3 sequence elsewhere. The results
reveal two regions in CTx ImI essential for binding to the
7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in
the first loop, where conservative mutations of each residue diminish
affinity by 2-3 orders of magnitude. The second region is the lone Trp
in the second loop, where an aromatic side chain is required. The
overall results suggest that within the triad of the first loop, Pro
positions the flanking Asp and Arg for optimal interaction with one
portion of the binding site, while within the second loop, Trp
stabilizes the complex through its aromatic ring.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Conotoxins are small, cysteine-rich peptides isolated
from the venom of marine cone snails (1). They competitively inhibit nicotinic acetylcholine receptors
(AChRs),1 and include various
subtypes which specifically target either muscle or neuronal AChRs. The
four cysteines in
-conotoxins form two intramolecular disulfide
bridges to produce a conformationally constrained two-loop structure
(Fig. 1). Muscle-specific
-conotoxins include MI, GI, and SI, while neuronal-specific toxins include ImI (CTx
ImI) and MII, which target
7 and
3
2 receptors, respectively (1-3). Owing
to their specificity for muscle and neuronal acetylcholine receptors,
-conotoxins are valuable probes of binding sites of nicotinic AChR
subtypes (4-7).
|
Specificity of a particular -conotoxin likely reflects structural
differences in the various AChR binding sites. Binding sites of
nicotinic AChRs are formed at interfaces between pairs of
and
non-
subunits (reviewed in Ref. 8). In the muscle AChR, the binding
sites are formed by
1-
,
1-
, and
1-
subunit pairs, whereas in the homomeric
7 AChR the binding sites are formed by pairs of
identical subunits,
7-
7. Thus across the various AChR subtypes, the different binding site interfaces contribute different residues which are recognized by the various
-conotoxins.
All -conotoxins contain two disulfide bridges, proline in the first
loop, and basic and aromatic residues in the second loop. However, each
-conotoxin targets a particular binding site through differences in
its number and type of residues. For example, CTx ImI contains four
residues in the first loop and three in the second, whereas
muscle-specific conotoxins contain three residues in the first loop and
five in the second. Furthermore, unlike muscle-specific conotoxins, CTx
ImI contains both positively and negatively charged residues in the
first loop. Thus structural differences in
-conotoxins likely
reflect structural differences at the various AChR binding site
interfaces.
Competitive antagonists are potential probes of binding site structure
that can identify residues of close approach which are distant in the
linear sequence or contained in different protein subunits. When the
antagonist is structurally constrained it can also serve as a molecular
caliper for estimating distances between these residues. For example,
previous work showed that the conformationally restricted antagonist
dimethyl-d-tubocurarine bridges the and
subunits in
the muscle receptor through interaction between its two quaternary
nitrogens and tyrosines in each subunit (9, 10). Given the distance
between quaternary nitrogens in dimethyl-d-tubocurarine, the
two tyrosines in the
and
subunits are estimated to be 11 Å apart. Similarly,
-conotoxins are potential probes of the binding
sites of muscle and neuronal AChRs. Solution and crystal structures of
-conotoxins reveal a triangular structure with positive charges at
two vertices separated by 15 Å (11-13). Thus, by identifying active
residues in CTx ImI and the
7 binding site interface,
residues of close approach can be identified, and their separation can
be estimated.
We recently identified residues of the 7 binding site
that confer neuronal specificity of CTx ImI (23). The present paper continues our work characterizing the
7 binding site by
constructing a series of CTx ImI mutants and measuring binding affinity
of the mutant toxins. The results reveal two key regions in CTx ImI essential for bioactivity.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
125I-Labeled -bungarotoxin
(
-Bgt) was purchased from NEN Life Science Products,
d-tubocurarine chloride from ICN Pharmaceuticals,
-conotoxins MI and GI from American Peptide Co., 293 human embryonic kidney cell line (293 HEK) from the American Type Culture Collection, and
-conotoxin SI, unlabeled
-Bgt, and DTNB
(5,5'-dithio-bis(2-nitrobenzoic acid)) from Sigma. Human
7 and rat 5HT-3 subunit cDNAs were generously provided by Drs. John Lindstrom and William Green. Sources of the human
acetylcholine receptor subunit cDNAs were as described previously
(15).
Synthesis and Purification of Conotoxin ImI--
Mutant and wild
type -conotoxin ImI were synthesized by standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems 431A peptide synthesizer. During synthesis, cysteine protecting groups (S-triphenylmethyl) were incorporated at
cysteines 3 and 12, and acetamidomethyl protecting groups (ACM) were
incorporated at cysteines 2 and 8. The linear peptide was purified by
reversed phase high performance liquid chromatography using a Vydac C18 preparative column with trifluoroacetic acid/acetonitrile buffers. The
two intramolecular disulfide bridges were formed as follows: the
cysteine S-triphenylmethyl protecting groups of cysteines 3 and 12 were removed during trifluoroacetic acid cleavage of the linear
peptide from the support resin, and the peptide was oxidized by
molecular oxygen to form the 3-12 disulfide by stirring in 50 mM ammonium bicarbonate buffer, pH 8.5, at 25 °C for
24 h. The peptide was lyophilized prior to formation of the second disulfide bridge. The ACM protecting groups on cysteine 2 and 8 were
removed oxidatively by iodine as described (16) except the
peptide/iodine reaction was allowed to progress 16 h prior to
carbon tetrachloride extraction. Residual iodine was separated from the
pure product by high performance liquid chromatography, and the product
was verified by mass spectrometry (Table I). The CTx ImI mutants are
named as follows: the first letter and number refers to the wild type
residue and position, and the following letter is the substituted
residue at that position.
Confirmation of Disulfide Bond Synthesis by Ellman's Analysis-- To confirm disulfide bond formation, we measured the colorimetric reaction of DTNB (Ellman's Reagent) with linear, non-oxidized CTx ImI and compared it with that of commercially available CTx MI and all of our synthetic CTx ImI mutants. 100 µg of each conotoxin was dissolved in 200 µl of 0.1 mM phosphate buffer, 4 µl of DTNB was added, the mixture was incubated at room temperature for 30 min for color development, and absorbance at 405 nm was measured. Reactivity of each synthetic CTx ImI mutant is expressed relative to that obtained for 100 µg of non-oxidized CTx ImI (Table I).
Construction of 7/5HT-3 Chimera and Expression in
293 HEK Cells--
Acetylcholine receptor subunit cDNAs were
subcloned into the cytomegalovirus-based expression vector pRBG4 (10).
The
7/5HT-3 chimera (
7200/5HT-3) was
constructed by bridging a 58-base pair synthetic oligonucleotide from a
TfiI site in human
7 to an EcoRV site in rat 5HT-3. The nucleotide sequence of the chimera was confirmed
by dideoxy sequencing. HEK cells were transfected with muscle or
7/5HT-3 cDNAs using calcium phosphate precipitation as described (10). Two days after transfection, intact cells were
harvested by gentle agitation in phosphate-buffered saline containing 5 mM EDTA for ligand binding measurements.
Ligand Binding Measurements--
Ligand binding to intact cells
was measured by competition against the initial rate of
125I--Bgt binding (10). The cells were briefly
centrifuged, resuspended in potassium Ringer's solution, and divided
into aliquots for ligand binding. Potassium Ringer's solution
contains: 140 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2,
25 mM HEPES, and 30 mg/liter bovine serum albumin, adjusted
to a pH of 7.4 with 10 mM NaOH. Specified concentrations of
ligand were added 30 min prior to addition of 3.75 nM
125I-
-Bgt, which was allowed to bind 15 min to occupy
approximately half of the surface receptors. Binding was terminated by
addition of 2 ml of potassium Ringer's solution containing 600 µM d-tubocurarine chloride. All experiments
were performed at 24 ± 2 °C. Cells were harvested by
filtration through Whatman GF-B filters using a Brandel Cell Harvester
and washed three times with 3 ml of potassium Ringer's solution. Prior
to use, filters were soaked in potassium Ringer's solution containing
4% skim milk. Nonspecific binding was determined in the presence of 10 nM
-Bgt and was typically 1% of the total number of
binding sites. The total number of binding sites was determined by
incubation with toxin for 120 min. The initial rate of toxin binding
was calculated as described (17) to yield the fractional occupancy of
competing ligand. Binding measurements were analyzed according to
either the monophasic Hill equation (Equation 1) or the sum of two
distinct binding sites (Equation 2),
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
7/5HT-3 Receptor Assay--
Studies of the
homomeric
7 binding site have been limited by low
expression of native
7 in mammalian cells (18). To
increase expression, we and others constructed
7/5HT-3
chimeras containing the extracellular domain of
7 joined
to the M1 junction of the rat 5HT-3 receptor (19, 20, 23). We showed
that the extracellular domain of human
7 maintained the
ligand recognition properties of wild type human
7, but
that the presence of 5HT-3 sequence greatly enhanced expression (23).
Thus, to determine affinities of the CTx ImI mutants described in the
present study, we measured binding to
7/5HT-3 receptors
expressed in 293 HEK cells.
Structures of Wild Type and Mutant CTx ImI-- Wild type and mutants of CTx ImI were synthesized as described under "Experimental Procedures." Molecular weights of each toxin were determined by mass spectrometry and compared with calculated molecular weights (Table I). The close agreement between measured and calculated molecular weights supports the amino acid compositions and formation of the two intramolecular disulfide bonds. To further confirm that both disulfide bonds formed, we assayed for free sulfhydryls using the colorimetric reaction of DTNB. Whereas the linear, nonoxidized CTx ImI reacted strongly with DTNB, neither the commercially available CTx MI nor any of the CTx ImI mutants reacted (Table I). Thus the mass spectrometry data combined with DTNB assay confirm that the wild type and mutant conotoxins are fully oxidized.
|
Neuronal Specificity of CTx ImI--
To establish that CTx ImI
shows the correct neuronal specificity for our 7/5HT-3
chimera, we compared binding of CTx ImI with that of the
muscle-specific conotoxins by competition against the initial rate
of 125I-labeled
-Bgt binding. As observed in other
expression systems (21), CTx ImI binds with much higher affinity to
7/5HT-3 receptors than CTx MI, GI, or SI (Fig.
2, top panel and Table
II).
|
|
Mutagenic Scan of CTx ImI--
We introduced conservative
substitutions for each non-cysteine residue in CTx ImI, and measured
binding of each mutant toxin to 7/5HT-3 receptors. The
results reveal two key regions in CTx ImI essential for high affinity
binding (Fig. 3 and Table
III). The first region is the triad
Asp-Pro-Arg in the first loop, where individual mutations decrease
affinity by 70-500-fold. The second region is the single tryptophan in
the second loop, which when mutated to threonine decreases affinity by
30-fold. On the other hand, mutating the four remaining non-cysteines
in CTx ImI does not significantly alter affinity for
7/5HT-3 receptors. These mutations include acetylation
of the amino-terminal glycine and neutralization of arginine in the
second loop (R11Q). Thus mutation of four of the eight non-cysteines in
CTx ImI alters affinity for
7/5HT-3 receptors.
|
|
Side Chain Specificity of the Active Residues--
To determine
the chemical nature of the contributions of each of the four essential
residues in CTx ImI, we introduced a systematic series of side chains
at each position and measured binding of each mutant toxin to
7/5HT-3 receptors (Fig.
4). Beginning with aspartic acid at
position 5, neutralization by substituting asparagine decreases
affinity by 100-fold, as described above (Fig. 3). However, replacement
with glutamic acid, which lengthens the side chain but maintains the
negative charge, decreases affinity even more. Introducing the
positively charged lysine produces the greatest decrease of affinity at
position 5. These results demonstrate that a negative charge and
correct side chain length are required at position 5, suggesting an
interaction with a focal electron acceptor in the receptor binding
site.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the basis of specificity of CTx ImI for neuronal
7 receptors, we used
7/5HT-3 chimeras to
express
7 binding sites and to measure binding affinity
of a series of CTx ImI mutants. The results reveal two regions of CTx
ImI that confer specificity for human neuronal
7
receptors. The first region is the conformationally sensitive triad
Asp-Pro-Arg within the first loop of CTx ImI. Subtle changes in side
chain lengths of the aspartic acid and arginine reduce affinity, and
their side chains appear to be held in place by the intervening
proline. Thus, the triad must maintain a specific conformation to fit
properly into a specific and focal counterpart in the
7
binding site. The second region is the single tryptophan within the
second loop of CTx ImI. Studies of side chain specificity at position
10 indicate the requirement of an aromatic ring.
Mutagenesis and site directed labeling studies establish that each
ligand binding site in muscle and neuronal AChRs contains contributions
from both and non-
subunits. Residues of the
portion of the
binding site, termed the (+) face, are located in three regions well
separated in the primary sequence, suggesting a three-loop model of the
(+) face of the binding site (reviewed in Refs. 8 and 22). Similarly,
residues of the non-
portion of the binding site, termed the (
)
face, are located in four separate regions of the primary sequence,
suggesting a four loop model for the (
) face of the binding site (8,
22). Unlike the two binding sites of the muscle AChR, which are formed
at interfaces between
1-
and either
1-
(fetal) or
1-
(adult) subunit
pairs, binding sites of the homo-oligomeric
7 receptor are formed at interfaces between pairs of identical subunits,
7-
7. Consequences of a homo-oligomeric
pentamer include the potential for five binding sites and formation of
both the (+) and (
) faces by a single
7 subunit.
-CTx ImI is a competitive antagonist of neuronal
7
receptors. It contains two disulfide bonds which hold the toxin in a constrained two-loop structure. The solution and crystal structures of
members of the
-conotoxin family reveal a compact triangular structure with two positive charges separated by 15 Å, similar to the
rigid structure and 11 Å separation of quaternary nitrogens of
curariform antagonists. Previous work showed that curariform antagonists bridge the interface between the
and
subunits of
the muscle receptor through quaternary-aromatic interactions (9).
Similarly, the two loops of CTx ImI likely bridge the (+) and (
)
faces of the
7 binding site. Because CTx ImI is small enough for structural determination at atomic resolution, it may be
used as a molecular caliper to estimate distances between points of
ligand contact at the
7 binding site.
Our studies reveal two distinct regions in CTx ImI essential for
binding to neuronal 7 receptors. The first region is
within the first loop of CTx ImI, the conformationally-sensitive triad Asp-Pro-Arg. Previous studies with CTx MI demonstrated the importance of proline in the first loop, where the mutation P6G reduced biopotency approximately 100-fold (14). Because our results with CTx ImI reveal a
similar decrease in affinity with P6G, and because Pro-6 is conserved
in all
-conotoxins, the proline likely contributes to structural
rigidity along with the two disulfide bridges.
Aside from Pro-6, no other essential residues have been reported in the
first loop of the -conotoxins. For CTx ImI, we show that aspartic
acid at position 5 and arginine at position 7 are essential for high
affinity binding. Surprisingly, conservative substitutions of Asp-5 and
Arg-7 markedly diminish activity of CTx ImI. The substitutions D5E and
R7K, which alter side chain length by one methyl group while
maintaining charge, reduce affinity more than 100-fold. In addition,
introducing opposite charges at Asp-5 and Arg-7 reduces affinity
approximately 1000-fold, suggesting repulsion by residues at the
7 binding site. The overall results of the Asp-5 and
Arg-7 side chain experiments suggest that both charge and side chain
length are important for the activity of CTx ImI. Together with residue
Pro-6, Asp-5, and Arg-7 form a conformationally sensitive triad
essential for CTx ImI affinity. Our results do not distinguish whether
P6 contributes directly to CTx ImI binding or structurally stabilizes
the first loop. However, the presence of a glycine mutation at position
6 likely allows rotational freedom around its
carbon that would
affect the orientation of the side chains of Asp-5 and Arg-7.
The second essential region in CTx ImI is the single Trp at position 10 of the second loop. Mutation of the two remaining non-cysteines in the
second loop fails to affect CTx ImI affinity. Similar to CTx ImI, the
muscle-specific conotoxins contain a conserved aromatic residue in the
penultimate position of the second loop (Fig. 1). Tryptophan of CTx ImI
occupies the position equivalent to that of tyrosine in CTx MI, GI, and
SI. Replacement of L-tyrosine with D-tyrosine
in CTx MI decreases bioactivity, indicating that the conformation of
the tyrosine is essential (14). Our results show that converting
tryptophan to threonine in CTx ImI reduces affinity 30-fold. However,
converting tryptophan to phenylalanine, which maintains the aromatic
side chain, decreases affinity only 3-fold. Thus an aromatic side chain
at position 10 stabilizes the CTx ImI-7 receptor
complex.
Surprisingly, mutations similar to those that affect affinity of muscle
-conotoxins do not affect affinity of CTx ImI. For example, a
cationic side chain in the second loop is critical for activity of the
muscle-specific
-conotoxins MI and SI (6, 7). Our results with CTx
ImI show no effect of the mutation R11Q. In addition, previous work
suggested that the N-terminal amide stabilizes the toxin-receptor
complex through a
-cation interaction (12). By contrast, acetylation
of the amino-terminal glycine does not affect affinity of CTx ImI for
7 receptors.
The overall results reveal two structural motifs in CTx ImI that confer
high affinity binding to 7 receptors. Knowledge of the
precise contacts between CTx ImI and
7 awaits
experiments that mutate residues in both the toxin and the receptor.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Denise Walker, Jane Liebenow, and Dr. Daniel McCormick of the Mayo Peptide Core Facility for synthesis of the CTx ImI mutants and Dr. William Green for providing the 5HT-3 cDNA.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant NS-31744 from the Mayo Foundation.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.
To whom correspondence should be addressed. Tel.: 507-284-5612;
Fax: 507-284-9420; E-mail: sine.steven{at}mayo.edu.
1
The abbreviations used are: AChR, acetylcholine
receptor; CTx ImI, -conotoxin ImI; 293 HEK, 293 human embryonic
kidney;
-Bgt,
-bungarotoxin; 5HT, 5-hydroxytryptamine; DTNB,
5,5'-dithio-bis(2-nitrobenzoic acid).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|