From the Department of Pharmacology 0636, University of California, San Diego, La Jolla, California 92093
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ABSTRACT |
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-Neurotoxins are potent inhibitors of the
nicotinic acetylcholine receptor (nAChR), binding with high affinity to
the two agonist sites located on the extracellular domain. Previous
site-directed mutagenesis had identified three residues on the
-neurotoxin from Naja mossambica mossambica
(Lys27, Arg33, and Lys47) and four
residues on the mouse muscle nAChR
-subunit (Val188,
Tyr190, Pro197, and Asp200) as
contributing to binding. In this study, thermodynamic mutant cycle
analysis was applied to these sets of residues to identify specific
pairwise interactions. Amino acid variants of
-neurotoxin from
N. mossambica mossambica at position 33 and of the nAChR at
position 188 showed strong energetic couplings of 2-3 kcal/mol at both
binding sites. Consistently smaller yet significant linkages of
1.6-2.1 kcal/mol were also observed between variants at position 27 on
the toxin and position 188 on the receptor. Additionally, toxin residue
27 coupled to the receptor residues 190, 197, and 200 at the
binding site with observed coupling energies of 1.5-1.9 kcal/mol. No
linkages were found between toxin residue Lys47 and the
receptor residues studied here. These results provide direct evidence
that the two conserved cationic residues Arg33 and
Lys27, located on loop II of the toxin structure, are
binding in close proximity to the
-subunit region between residues
188-200. The toxin residue Arg33 is closer to
Val188, where it is likely stabilized by adjacent negative
or aromatic residues on the receptor structure. Lys27 is
positioned closer to Tyr190, Pro197, and
Asp200, where it is likely stabilized through electrostatic
interaction with Asp200 and/or cation/
interactions with
Tyr190.
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INTRODUCTION |
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The nicotinic acetylcholine receptor
(nAChR)1 is a member of the
large family of neurotransmitter-gated ion channels (for review, see
Refs. 1-4). It is composed of five subunits that are arranged in the
circular order of . The two ligand binding sites reside in the extracellular domain at the
and
subunit interfaces (5, 6). A high resolution structure is not available for any member of
the family of neurotransmitter-gated ion channels. However, within this
family the structure of the nAChR has been most extensively
characterized through site-directed labeling (7-12), site-specific
mutagenesis (13-23), electron microscopy reconstruction analysis (24,
25), and homology modeling (26). In current models, three discontinuous
regions or domains on the
-subunit (encompassing residues around 93, 149-154, and 180-200) and four regions on the
/
subunits are
thought to participate in the formation of the binding sites (2, 26).
Because of sequence differences in the
and
subunits, the
binding sites on the receptor are not identical; consequently, many
ligands bind to each site with different affinities (15, 22, 27).
The critical tool utilized in the initial identification of the
receptor and in subsequent structural analyses is the family of
three-fingered snake -neurotoxins (for review, see Refs. 28 and 29),
which form high affinity complexes with the receptor; for example, the
-bungarotoxin-receptor complex has a Kd of
~10
12. The structure of the
-neurotoxins has been
solved through nuclear magnetic resonance (30-32) and x-ray
crystallographic studies (33-35). These polypeptides (~7 kDa) are
characterized by three large loops which extend from a rigid globular
domain held together by 4 or 5 conserved disulfide bonds. Even though
-neurotoxin structures have been solved, little is known about the
structure of the toxin-receptor complex, and interacting residues have
not been identified.
In previous work, we delineated residues involved in the binding
interaction on both the -neurotoxin, Naja mossambica
mossambica (NmmI), and the mouse muscle nAChR interfaces (36).
Four residues located on the receptor
-subunit and three residues
located on the toxin structure were found to contribute significantly
to high affinity binding. Even though wild-type NmmI displays an equivalent affinity for the two binding sites on nAChR, we showed through mutational analysis that the energetic contribution of selected
residues differed at the two sites. Several of the toxin and receptor
mutations studied differentially affected binding at the
and
sites, resulting in two distinct binding affinities (36). In
this study, we have utilized double mutant cycles to explore whether
any of these residues are involved in pairwise interactions at each
binding site. Such information may not only lead to a better
understanding of the structure of the toxin-receptor complex, but
combined with the known structure of the toxin may eventually
establish spatial constraints within the receptor
architecture (37-39).
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EXPERIMENTAL PROCEDURES |
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Materials--
125I--Bungarotoxin (specific
activity ~16 µCi/µg) was obtained from NEN Life Science Products.
-Conotoxin M1 was purchased from American Peptide Company.
NmmI Expression and Purification--
Recombinant wild-type and
mutant NmmI were expressed as fusion proteins in Escherichia
coli, and the free toxins were purified as described (36). Because
several of the mutant toxins produced rather low affinity complexes
with mutant receptors, relatively large amounts of NmmI -neurotoxins
were required for this study. Typically, 0.5-1.5 mg of toxin could be
purified to homogeneity from 1 liter of cells.
Receptor Mutagenesis-- All receptor mutants were made as described previously (20, 23, 27, 36, 40).
Expression of Wild-type and Mutant nAChR--
cDNAs encoding
the mouse muscle nAChR subunits (,
,
, and
) in a
cytomegalovirus-based expression vector pRBG4 were co-transfected in a
ratio of 2:1:1:1 into HEK 293 cells (at ~50% confluency) using
Ca3(PO4)2 precipitation. After
16 h, the medium containing cDNA was replaced with fresh
medium (Dulbecco's modified Eagle's medium plus 10% fetal calf
serum), and expression was measured 3-4 days after transfection
(20).
NmmI Binding Measurements--
Binding assays were carried out
on assembled pentameric nAChRs expressed on the surface of intact
cells. The cells were harvested by gentle agitation in
phosphate-buffered saline plus 5 mM EDTA, centrifuged
briefly, and resuspended in high potassium Ringer's solution. The
cells were divided into aliquots for binding measurements (assay volume
200 µl). Specified concentrations of NmmI were added to each tube
containing receptor and allowed to bind for 5 h. NmmI dissociation
constants were measured by competition against initial rates of
125I--bungarotoxin binding using 10-20 nM
concentrations (41).
Homology Modeling--
Using erabutoxin b as a template (an
-neurotoxin of known crystal structure (33) and possessing 60%
residue identity with NmmI), segments of NmmI were modeled on the basis
of conserved regions and the common disulfide linkages. The final
conformation of the
-carbon backbone was adjusted to account for the
two unique prolines in erabutoxin b and the single proline in NmmI. The
modeled structure was then relaxed by unrestrained steepest descent
minimization of 5000 iterations with the program Discover (MSI, 1997).
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RESULTS |
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nAChR Binding Site--
Of the three segments of linear sequence
in the -subunit, the region encompassing residues 180-200 appears
to be the most critical for NmmI recognition. Substitutions at residues
188, 190, 197, and 200 resulted in substantial decreases in toxin
binding affinity (36), indicating major roles of these residues in
-neurotoxin recognition. Determinants in this region include a
conserved aromatic residue (Tyr190), two positions where
neuronal and muscle receptors differ (Val188 and
Pro197), and a negatively charged residue
(Asp200). The mutations Y190F and Y190T both resulted in a
loss of affinity of ~2 kcal/mol at the
site and almost 4 kcal/mol at the
site (Table I),
suggesting the importance of the aromatic hydroxyl group. Introducing a
positive charge at position 188 (V188K) was also unfavorable and
destabilized the toxin-receptor complex by 1.8 kcal/mol (at the
site) and 3.5 kcal/mol (at the
site), whereas introduction of a
negative charge (V188D) at this position had either no effect or far
less of an influence (
0.09 kcal/mol at the
site and 1.8 kcal/mol at the
site). The selective loss in binding affinity
observed with the introduction of a positive charge but not a negative
charge suggested destabilization resulted from coulombic repulsion of
the highly cationic
-neurotoxin. Elimination of the negative charge
at position 200 (D200Q) decreased binding by 66-fold (2.5 kcal/mol)
selectively at the
site.
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-Neurotoxin Structure and Binding Site--
Sequence
comparisons between members of the
-neurotoxin family have
identified ~12 residues that are highly conserved, three of which are
positively charged (Lys27, Arg33,
Lys47; NmmI numbering). Extensive mutagenesis studies
carried out by Menez and co-workers (42-44) using a homologous
-neurotoxin with 60% residue identity to NmmI (erabutoxin a)
defined the toxin binding surface to encompass ~680 Å2
and about 10 amino acids located on loop II and the tips of loops I and
III. Five of these residues appear to play the predominant role in
binding (Ser8, Gln10, Lys27,
Arg33, Lys47) (Fig.
1). The binding site utilized for the
NmmI toxin appears to be similar yet not identical to that defined for
erabutoxin a (36). Whereas Gln10 in erabutoxin a and
Glu10 in NmmI affect binding differently, similar to
erabutoxin a, the three conserved positive residues are critical for
the NmmI-mouse muscle nAChR high affinity interaction.
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Double Mutant Cycles--
Double mutant cycle analyses were
applied to the three -neurotoxin variants (K27E, R33E, and K47A) and
six
-subunit nAChR variants (V188D/K, Y190T/F, P197I, and D200Q)
described in order to delineate potential pairwise interactions. This
method is based on simple additivity or non-additivity of mutations. If
two residues are interacting, then the sum of the free energy
change of the single mutations will usually not equal the free
energy change measured with both mutations (45-47). This is shown by
Equation 1:
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
Mutant Pairs and Site Selectivity--
Dissociation constants and
changes in free energy for each of the mutant pairs analyzed are shown
in Table I. Fig. 2 (A and B), shows typical binding curves obtained with the
-subunit mutations Y190T and P197I, respectively, when assayed with
each of the toxin mutations; the wild-type toxin/wild-type receptor
curve is also shown for comparison. As seen in Table I and Fig. 2, many
of the mutant pairs result in large reductions in the overall affinity of the toxin-receptor complex. The dissociation constant for the R33E/V188D pair at the
binding site was too large to measure precisely but corresponded to a Kd of more than 0.5 mM. The R33E/P197I mutant pair resulted in a loss of
affinity of 8.0 kcal/mol (
site) or a loss in
Kd of 6 orders of magnitude compared with the
wild-type/wild-type interaction. Mutant pairs involving the toxin
mutation K27E also resulted in large destabilizations ranging from 3.5 to 7.5 kcal/mol.
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Coupling Analysis--
The coupling coefficient and coupling
energy
GINT for each toxin-receptor pair
(at both the
and
binding sites) were determined using
Equations 1 and 2 (Table I and Fig. 3).
Two of the mutant pairs studied gave strong coupling energies above 2.0 kcal/mol and seven pairs gave coupling energies of 1.5 kcal/mol or
above. Despite very large shifts in the overall affinities of the
mutant pairs relative to that of the wild-type toxin-wild-type receptor
complex, the majority of the pairs showed simple additivity of free
energy within experimental error (coupling energies approaching 0, 0.1-0.5 kcal/mol). Most strikingly, all of the mutant pairs involving
K47A gave values very close to
GINT = 0, indicating that the introduced receptor mutations and the toxin
mutation K47A do not grossly alter the respective protein structures. A few mutant pairs gave values between 1.0 and 1.3 kcal/mol; intermediate energy values in this range are difficult to interpret because of
cumulative errors of addition of free energies.
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Interactions of Receptor Residue Val188 with Toxin
Residues Arg33 and Lys27--
The two
strongest interactions were found between the mutant pairs R33E/V188D
and K27E/V188D at the interface (coupling energies of 2.6 and
2.1 kcal/mol, respectively). The R33E/V188D pair also appeared to have
a strong coupling at the
interface (>1.5 kcal/mol), but a
precise number could not be obtained. The very large overall loss in
the binding affinity required an unachievable production level of
mutant toxin. On the other hand, no significant coupling was observed
at the
interface with the K27E/V188D mutant pair containing the
other charge substitution studied in loop 2.
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Other Interactions--
The toxin-receptor mutant pairs
K27E/Y190F, K27E/Y190T, K27E/P197I, and K27E/D200Q all showed coupling
energies of 1.5 kcal/mol or higher at the binding site with no
significant couplings observed at the
binding site.
Approximately equal coupling values were found with either Y190T or
Y190F and the K27E substitution (Table I). Apart from its coupling to
position 188 on the receptor, the R33E substitution showed coupling to
the Y190T mutation. This was only observed at the
binding
site.
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DISCUSSION |
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Using site-directed mutagenesis, we previously identified residues
involved in the high affinity interaction between the -neurotoxin NmmI and the mouse muscle nAChR. The goal of this study was to identify
specific pairs of interacting residues between the toxin and the
receptor utilizing double mutant cycles (39, 47). This method has been
successfully developed to identify pairwise interactions between the
scorpion toxins and the potassium channel (37-39). In those studies,
the known structure of the toxin was utilized to establish spatial
locations of residues on the potassium channel of unknown
structure.
In theory, if two residues are coupled either directly or through
another residue then the effect of the double mutation will not be
equal to the sum of the effects of the two single mutations (47, 48).
Therefore, if GINT deviates from 0 (or
deviates from unity) the two mutations under study exhibit an
interaction. In practice, small
GINT
values may exist between residues separated by great distances (49).
Conversely, coupling energies greater than 1.5 kcal/mol are generally
associated with short distances between the two residues under study
(46). In our case, the errors associated with binding measurements of
two nonequivalent sites located on one receptor molecule ranged from 10 to 20%. Because of the cumulative errors when summing single
mutations, the errors associated with our linkage values ranged from 30 to 50%. Therefore, we have only considered coupling energies above 1.5 kcal/mol for our analysis.
Besides direct interactions or interactions mediated through a proximal residue, large deviations from additivity can also occur when gross structural changes in the individual molecules result from the introduced mutations. If a mutation results in a global conformational change then it would be expected to be linked to a large number of residues. Therefore, the lack of linkages with nearby residues becomes a good indication that structural integrity of the interacting molecules has been maintained. In addition, failure to detect an interaction does not exclude the close proximity of the two residues. This may be due to either weak interactions between the two residues or to interactions that are compensatory yielding a minimal net change. This is especially true in the case of mutant cycles in which the reference side chain, typically the naturally occurring amino acid, exhibits a dominant influence (50).
For example, when the naturally occurring side chain Val188
on the common -subunit of two binding sites is converted to both cationic and anionic side chains therein creating two parallel cycles,
different coupling energies are achieved at the
and
interfaces (Fig. 4). However, if we coalesce the two cycles by
considering a direct substitution from a cationic to an anionic side
chain, then differences between the two sites virtually vanish. This
suggests the naturally occurring valine common to both small cycles
imparts the asymmetry, and the influence of an inserted charge on
-neurotoxin binding is similar at both sites. Interactions intrinsic
to the reference residue or steric constraints could influence the
and
sites in a differential manner (50). A more
appropriate frame of reference might come from a neutral side chain
isosteric with substituted residues or a side chain with minimal steric
perturbation (alanine).
Of the 36 residue pairs studied here (18 at each of the and
binding sites), 25 gave
G values below 1.0 kcal/mol, indicating simple additivity. These results suggest that the
gross structural changes do not occur in the interacting molecules with
the introduced mutations. Rather, the relatively few but large coupling
energies that were observed support the specific interactions between
these residues.
The strongest linkages observed are with the R33/V188 and K27/V188
toxin-receptor pairs. The strength of the coupling observed between
these pairs varied at the and
binding sites and also with
the different amino acid substitutions examined. The toxin residue 33 and receptor residue 188 appear to be interacting at both the
and
binding sites with coupling energies as high as 2.8 and
>3.0 kcal/mol observed within the network of cycles. Toxin residue 27 and receptor residue 188 also appear to be interacting at both sites
but to a lesser extent, as the coupling energies observed between this
pair were generally lower than with the 33/188 pair. In contrast, the
toxin residue Lys47 did not show any interaction with the
receptor residue Val188.
Coupling energies ranging from 1.5 to 1.9 kcal/mol were also found with
the toxin residue Lys27 and the three receptor residues
(Tyr190, Pro197, and Asp200), all
at the interface. These results suggest that Tyr190,
Pro197, and Asp200 are close enough in the
receptor structure each to be interacting with Lys27.
Another possibility is that some or all of these observed linkages are
mediated through a third residue. The lack of coupling observed between
these paired residues at the
binding site does not preclude
their interaction or their close proximity. However, it does
demonstrate that the energetic contributions of Tyr190,
Pro197, and Asp200 to toxin binding differ at
the two sites. These data also suggest that the toxin is binding with
different orientations to the two ligand sites, where interactions with
Tyr190, Pro197, and Asp200 are less
critical for the
site, but further experiments will be necessary
to address this point.
An initial model of binding is proposed from these data. The two
conserved toxin cationic residues Arg33 and
Lys27, located on loop II of the toxin structure, are
complexing with key receptor residues located on the -subunit region
between 180 and 200. More specifically, we suggest that the toxin
residue Arg33 is adjacent to the receptor residue
Val188 and is probably stabilized by adjoining negative or
aromatic residues located on the receptor structure. One such candidate may be Tyr190, which did show a linkage with
Arg33 at the
binding site. Other possibilities
include residues located on the
/
subunits (see below).
Lys27 also appears to be positioned in the vicinity of
Val188 but closer to the residues Tyr190,
Pro197, and Asp200. In this case, the lysine
cation on the toxin may be directly stabilized through electrostatic
interactions with Asp200 and cation/
interactions with
Tyr190.
The involvement of cationic residues near the tip of loop 2 on the
toxin and the receptor sequence between residues 180 and 200 has been
implicated from single residue mutations, chemical labeling, and
binding of toxin to receptor peptide fragments (50-55), but previous
studies have not pinpointed specific residue interactions nor have they
distinguished differences in -neurotoxin binding between the two
binding sites. Homology modeling (26) and labeling experiments (12)
have indicated that residues 180-200 on the
-subunit are located at
the interface formed at the
and
ligand binding sites.
Therefore, besides the
-subunit residues studied here, it is likely
that the toxin loop II residues are interacting with
/
subunit
residues. On the other hand, K47A, which is located on loop III of the
toxin structure, does not appear to be interacting with this area of
the
-subunit. Further studies aimed at identifying linkages between
toxin residues and receptor residues on the
and
subunits should
provide the additional constraints necessary to describe the toxin
orientation and positioning at the two receptor binding sites.
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ACKNOWLEDGEMENTS |
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We thank Francisco Lio for technical
assistance with the binding assays and Dr. David A. Johnson (University
of California, Riverside) for providing cobra -toxin.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Fellowship NS 10082 (to E. J .A.) and United States Public Health Service Grant GM 18360 (to P. T.).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: Dept. of
Pharmacology 0636, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 619-534-1366; Fax:
619-534-8248; E-mail: priley{at}ucsd.edu.
1
The abbreviations used are: nAChR, nicotinic
acetylcholine receptor; NmmI, -neurotoxin I from Naja
mossambica mossambica.
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REFERENCES |
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