Subunit Interface Selectivity of the
-Neurotoxins for the
Nicotinic Acetylcholine Receptor*
Hitoshi
Osaka
,
Siobhan
Malany§,
Joan R.
Kanter,
Steven M.
Sine¶, and
Palmer
Taylor
From the Department of Pharmacology 0636, University of California,
San Diego, La Jolla, California 92093, and the ¶ Receptor
Biology Laboratory, Department of Physiology and Biophysics, Mayo
Foundation, Rochester, Minnesota 55905
 |
ABSTRACT |
Peptide toxins selective for particular subunit
interfaces of the nicotinic acetylcholine receptor have proven
invaluable in assigning candidate residues located in the two
binding sites and for determining probable orientations of the bound
peptide. We report here on a short
-neurotoxin from Naja
mossambica mossambica (NmmI) that, similar to other
-neurotoxins, binds with high affinity to 
and 
subunit
interfaces (KD~100 pM) but binds with
markedly reduced affinity to the 
interface
(KD~100 nM). By constructing chimeras
composed of portions of the
and
subunits and coexpressing them
with wild type
,
, and
subunits in HEK 293 cells, we
identify a region of the subunit sequence responsible for the
difference in affinity. Within this region,
Pro-175 and
Glu-176 confer high affinity, whereas Thr and Ala, found at
homologous positions in
, confer low affinity. To identify an
interaction between
Glu-176 and residues in NmmI, we have examined
cationic residues in the central loop of the toxin and measured binding
of mutant toxin-receptor combinations. The data show strong pairwise
interactions or coupling between
Glu-176 and Lys-27 of NmmI and
progressively weaker interactions with Arg-33 and Arg-36 in loop II of
this three-loop toxin. Thus, loop II of NmmI, and in particular the
face of this loop closest to loop III, appears to come into close
apposition with Glu-176 of the
subunit surface of the binding site interface.
 |
INTRODUCTION |
The nicotinic acetylcholine receptor
(nAChR)1 found in muscle is a
pentamer composed of four homologous subunits present in the
stoichiometry
2

(fetal subtype) or
2

(adult subtype). The subunits are arranged in
a circular manner to surround a central channel in the order,




or 



(1-3). The two binding sites for
agonists, competitive antagonists, and the slowly dissociating
-neurotoxins are formed at interfaces between the 
and

(
) subunit pairs. The extracellular domain in each subunit is
formed principally from the amino-terminal 210 amino acids, which is followed by four membrane-spanning domains. Residues within the amino-terminal 210 amino acids have been shown to be the major contributors to the ligand binding sites and for dictating the order of
assembly of subunits.
Three segments of the
subunit, well separated along the linear
sequence, harbor major determinants for ligand binding; these segments
contain the key residues around Tyr-93, between Trp-149 and Asp-152,
and spanning the region from Val-188 through Asp-200 (see Refs. 3 and 4
for reviews). Similarly, four discontinuous segments of the non-
subunits, appearing on the opposite face of the subunit, contain major
determinants for ligand selectivity; in the
subunit these segments
contain the key residues Lys-34, between Trp-55 and Gln-59, between
Ser-111 and Tyr-117, and between Phe-172 and Asp-174.
Since the early demonstration of irreversible neuromuscular blockade by
the peptide from snake venom,
-bungarotoxin (5), and the use of
labeled
-neurotoxins to identify the nAChR (6), these toxins have
been the primary ligands employed for the identification and
characterization of the muscle nAChR. Amino acid sequences are
available for nearly 100 members of the
-neurotoxin family, which
show a common basic structure consisting of three polypeptide loops
emerging from a small globular core (7).
-Neurotoxins can be divided
into the short (4 disulfide bonds and 60-62 residues) and long
neurotoxins (5 disulfide bonds and 66-74 residues). Crystal and
solution structure determinations reveal similar tertiary structures.
Although these structurally well defined toxins are known to bind at
the subunit interfaces (
or 
and 
), typically with a
KD
100 pM, little is known about
their precise orientation with respect to the subunits that form the interfaces.
Points of attachment of
-neurotoxin within the nAChR binding sites
have been examined by cross-linking chemically modified (8, 9) or
photoactivatable derivatives of
-neurotoxin (10-13) and by simple
ultraviolet irradiation without chemical modification (14). These
labeling studies have suggested contacts with both
and non-
subunits at the binding sites (see Refs. 2, 3, and 15 for reviews).
Mutagenesis studies have also identified candidate residues in the
principal loops of the
(16) and non-
subunits (17) that
contribute to
-toxin binding. Although most
-toxins do not
distinguish between the two sites on the receptor, an
-toxin from
the venom of Naja mossambica mossambica (NmmI) distinguishes
between the two sites of the Torpedo receptor (18). Thus
NmmI emerges as a potentially valuable ligand for determining regions
of close approach between
-toxins and the non-
subunits at the
binding site. Previous work showed that the 
and 
binding
sites of the fetal mouse receptor exhibit similar affinities for NmmI
(19). However, certain mutations in the NmmI toxin structure, and
surprisingly also in the nAChR
subunit common to both sites,
resulted in nonequivalent reductions in affinity at the 
and

binding sites (19). Here we examine binding of recombinant NmmI
-toxin to fetal and adult mouse AChRs and find that the affinity of
NmmI for the 
interface is 3 orders of magnitude lower than for
the 
and 
interfaces. Using subunit chimeras and
site-directed mutations in
and
subunits, we show that the
enhanced affinity conferred by the
over the
subunit arises from
Pro-175 and Glu-176 in the
subunit. Mutant cycle analysis shows
that Glu-176 interacts with cationic residues in loop II of the NmmI
-toxin.
 |
EXPERIMENTAL PROCEDURES |
Materials--
-Conotoxin MI was purchased from American
Peptide Company. 125I-labeled
-bungarotoxin (
-BgTx)
(specific activity ~16 µCi/µg) was a product of NEN Life Science Products.
NmmI Expression and Purification--
A double-stranded
synthetic NmmI cDNA containing the
-erabutoxin signal sequence
and strategically placed restriction sites was subcloned into pEZZ
vector encoding two IgG binding proteins from Staphylococcal protein A. The Staphylococcal protein A-NmmI fusion protein was expressed using
Escherichia coli HB 101, cleaved, and purified as described
in Ackermann and Taylor (19).
Construction of Mutant nAChR--
cDNAs encoding mouse nAChR
subunits were subcloned into a cytomegalovirus-based expression vector,
pRBG4. All mutations were introduced using Quick ChangeTM
Site-Directed Mutagenesis Kit (Stratagene) or by bridging two introduced or natural restriction sites with double-stranded
oligonucleotides. Chimeras were also constructed by bridging natural or
introduced restriction sites. After ligation of the fragments
containing the mutated site or synthesized oligonucleotide into the
original pRBG4 vector, the subcloned cassette was sequenced by the
dideoxy method.
Cell Transfections--
cDNAs encoding the wild type and
mutant subunits were transfected into human embryonic kidney (HEK 293)
cells using Ca3(PO4)2 in the
ratios:
(15 µg)/
(7.5 µg)/
or
(7.5 µg)/
(7.5 µg).
Ligand Binding Measurements--
Cells were harvested in
phosphate-buffered saline, pH 7.4, containing 5 mM EDTA,
2-3 days after transfection. They were briefly centrifuged,
resuspended in potassium-Ringers buffer, and divided into aliquots for
binding assays. Specified concentrations of NmmI and conotoxin MI were
added to the samples 20 min prior to initiating the association rate
assay with 125I-labeled
-bungarotoxin. Dissociation
constants of the ligands were determined from their fractional
reduction of the initial rate of 125I-labeled
-bungarotoxin association (20, 21). Appropriate concentrations of
-conotoxin MI were used to block the 
interface but not that
of 
, when the distinctions in affinity for the 
and 
interfaces were not obvious (19).
Rate Measurements--
The association rate for
125I-labeled
-bungarotoxin was measured using a
-bungarotoxin concentration of 20 nM. At the specified time, cells were washed with 30 mM carbamylcholine in
K+-Ringers solutions and then washed two times with
K+-Ringers solution alone and counted. For the measurement
of dissociation rates, we equilibrated the surface receptor with 40 nM of 125I-labeled
-bungarotoxin for 2 h, then washed the cells two times with K+-Ringers to
remove the unbound ligand and resuspended the cells in
K+-Ringers with a 10-fold dilution. Unbound toxin was
removed by washing at specific times, and the cells were counted
(16).
 |
RESULTS |
Insensitivity of the 
Site to NmmI--
Certain competitive
antagonists distinguish between the two binding sites of the nAChR
because of species or subtype differences in the non-
subunits that
form the 
, 
, and 
binding sites. We therefore
compared binding of NmmI to mouse fetal (
containing) and adult (
containing) nAChRs expressed in HEK 293 cells. As described previously,
NmmI does not distinguish between 
and 
sites of fetal
receptors, binding to a single class of sites with a
KD of 0.14 nM (Ref. 19; Fig.
1). Surprisingly, however, NmmI selects
strongly between 
and 
sites of the adult receptor, binding
to the 
site with three orders of magnitude lower affinity
(KD = 130 nM; Fig. 1). Thus the
subunit of the adult receptor contains residues that confer
insensitivity to NmmI.

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Fig. 1.
Sensitivity of nAChR expressed as
2 
and
2 
in HEK cells to -neurotoxin from
Naja mossambica mossambica
(NmmI). HEK cells were transfected
with cDNAs encoding wild type subunits to form cell surface
receptors with the composition 2  ( ) and
2  ( ). -Neurotoxin (NmmI) binding was
measured by competition with an initial rate of -bungarotoxin
binding. kobs/kmax is the ratio of initial rates for
125I-labeled -bungarotoxin binding in the presence and
absence of NmmI. Data are plotted according to the equation:
kobs = 0.5 kT, ([NmmI]/([NmmI] + K )) + 0.5 KT, ([NmmI]/([NmmI] + K )), where
kT, and kT,
are the -bungarotoxin association rates for the  and 
sites, K and
K are the equilibrium dissociation
constant for NmmI of the respective sites, and
kmax = 0.5 kT, + 0.5 kT, . For 2  ,
the data are fit to a single class of sites. The fraction of sites with
an affinity corresponding to  and  is reflected by the
inflection. The apparent fraction of sites of  (0.62) to 
(0.38) in 2  that is greater than the predicted
equal population of sites since kT, > kT, (cf. Fig. 2).
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Measurements of NmmI binding also show apparently unequal fractions of
high and low affinity sites in the adult receptor (fraction of high
affinity sites = 0.63 ± 0.03; n = 3), rather
than a value of 0.5 expected for an equal abundance of 
and

sites. This apparent inequality could arise from distinct rates
of association of the reporter ligand 125I-labeled
-bungarotoxin with 
and 
sites. To examine this possibility, we compared time courses of association of
125I-labeled
-bungarotoxin for fetal and adult nAChRs
(Fig. 2). For the
2

fetal receptor, association is well described
by a single exponential component with a kon of
3.6 ± 0.7 × 106 M
1
min
1(n = 5), indicating indistinguishable

and 
sites. On the other hand, for the
2

adult receptor, association can be described by two exponential components with equal amplitudes, with a faster component of kon = 3.9 ± 0.5 × 106 M
1 min
1 and
slower component of kon = 1.0 ± 0.2 × 106 M
1
min
1(n = 2). The different association
rates could account for the apparently unequal fractions of sites
detected by NmmI when it competes with the initial rate of
125I-labeled
-bungarotoxin binding (17). The apparent
fraction of the two sites will be biased toward the site with the more rapid rate of
-bungarotoxin binding.

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Fig. 2.
Kinetics of
125I-labeled
-bungarotoxin association with the nAChR expressed
as
2 
and
2 
in HEK cells. Top panel, association of 5 nM 125I-labeled -bungarotoxin with 200 pM wild type 2  ( ) and
2  ( ) receptors. The data for
2  are fit by a single exponential approach to
equilibrium with a kon of 3.6 ± 0.7 × 106 M 1 min 1,
whereas for 2  a two-exponential fit of equal
amplitudes is used kon of 3.9 ± 0.5 × 106 M 1 min 1 and
1.0 ± 0.2 × 106 M 1
min 1. Bottom panel, dissociation of
125I-labeled -bungarotoxin after equilibration of 40 nM toxin with 20 pM receptor, washing twice
with K+-Ringers solution, and resuspending with dilution in
K+-Ringers solution. Averaging three such dissociation
experiments yields koff = 2.5 ± 0.5 × 10 4min 1 for 2 
and 2.6 ± 0.6 × 10 4min 1 for
2  .
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We also compared time courses of dissociation of
125I-labeled
-bungarotoxin from fetal and adult
receptors. The two types of receptors did not show significant
differences in dissociation time courses as estimated from the initial
portion of the dissociation profiles with koff = 2.5 ± 0.5 × 10
4 min
1 for
2

and koff = 2.6 ± 0.6 × 10
4 min
1 for
2

(n = 3). The slow rates of
dissociation preclude measurements over the entire time course because
of progressive autolysis of the preparation. The ratios of dissociation
to association rate constants yield equilibrium dissociation constants
for
-bungarotoxin of 69 pM (
and 
) for
2

and 67 pM (
), 260 pM (
). These kinetic experiments demonstrate that
-bungarotoxin, has far less capacity than NmmI to distinguish
between the 
and 
sites.
Molecular Basis of Insensitivity of the 
Site for NmmI
Toxin--
The
and
subunits show high sequence identity in the
extracellular domains (54% in mouse), and homologous residues should have virtually identical locations for their
-carbon backbone positions. Yet, NmmI binds 1000-fold more tightly to the 
than to
the 
site. To determine the structural basis of NmmI selectivity, we constructed subunit chimeras containing portions of the
subunit substituted into the
subunit. Each chimera was coexpressed with complementary
,
, and
subunits, followed by measurements of NmmI binding. We first screened with chimeras containing
sequence from the amino terminus to junctions ranging from positions 74 to 173 of the
subunit. Each of these chimeras confers low affinity for
NmmI, characteristic of the wild type 
site (Fig.
3), indicating that NmmI selectivity
arises from residues carboxyl-terminal to position 173. By contrast,
moving the chimera junction just four residues to position 177 increases NmmI affinity to that of the native 
site
(KD = ~80 pM; Fig. 3).
Thus residue differences between
positions 174 and 177 confer NmmI selectivity for the 
over the

site.

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Fig. 3.
NmmI -toxin
association with the nAChR formed by transfection of cDNAs encoding
chimeras of the and subunits, along with ,
, and subunits.
Left, schematic representation of chimeric cDNAs formed
between the and subunits. ( 74 ) denotes that the
amino-terminal 74 amino acids are constructed from subunit
(shaded), and residues carboxyl-terminal to this position
come from subunits (nonshaded). M1-4 denotes the
putative transmembrane regions. Right: The dashed vertical line
represents the KD for NmmI binding to the wild type
 interface. The dashed bars show the actual
KD for each chimera (top ruler) and the
log
[KD,mt/KD,wt
] (bottom ruler).  G values can be obtained
from multiplying by 2.3 RT.
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Table I
The influence of -toxin and receptor mutations at the , , and
subunits on Naja mossambica mossambica (NmmI) -toxin association
with the nicotinic acetylcholine receptor
Dissociation constants were calculated from competition with the
initial rate of the 125I-labeled -bungarotoxin binding.
Receptor was expressed as 2  or
2  by transfection of cDNAs encoding four
respective sets of subunits. KD is dissociation
constant for  ,  , or  sites by fitting a two-site
analysis. The ratios of dissociation constants of mutant (mt) to wild
type (wt) were calculated using an average or mean value of at least
two measurements involving separate transfections.  G
is free energy of binding calculated from Equation 1 in the text.
 GINT was calculated using Equation 2 in the
text. Values less than unity were inverted and indicated with a minus
sign.
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Sequence comparison of the
and
subunits reveals only two
mismatched residues between positions 174 and 177 (Fig.
4A). We therefore constructed
point mutations at these two positions of the
subunit and measured
NmmI binding to the resulting mutant receptors. The point mutations
P175T and
E176A reduce affinity to values intermediate to those
of the wild type 
and 
sites (P175T,
KD,mt/KD,wt = 36; E176A,
KD,mt/KD,wt = 16; Fig. 4, Table
I). Combining the two mutations into a single
subunit reduces
affinity to approach that of the wild type 
site (Fig. 4, Table
I). The converse double mutation in the
subunit,
T176P and
A177E, increases NmmI affinity to equal that of the wild type 
site (Fig. 4, Table I). Thus, the residue pairs at homologous positions
Pro-175/
Thr-176 and
Glu-176/
Ala-177 fully account for the
1000-fold selectivity of NmmI for the 
over the 
site.

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Fig. 4.
Assignment of the residues contributing
to subunit insensitivity to NmmI
binding. A, top, the junctions of two
chimeras, 173 and 177 . The shaded bar represents
subunit sequence followed the nonshaded subunit. Junctional
amino acids are shown by the bar. Amino acids from are
designated in italics. Numbering is for subunit. Only two
differences at positions 175 and 176 ( 176/177) exist in this
region. A, bottom, mutations (*) were introduced
at or subunits to examine the contributions at these two
positions. KD and the
KD,mt/KD,wt
are shown in a logarithmic scale as described in Fig. 2. B,
sequence alignment around 175 and 176 in the subunit. The
superscripts in italics show reported determinants for binding of
various ligands. Superscripts are: waglerin, W (30);
-conotoxin MI, CM (29); anionic residues for agonists,
A (25, 26), and acetylcholine, ACh (36).
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Sequence alignment shows that the
subunit contains the same
residues in this region as the
subunit, consistent with high affinity of the 
site. We therefore attempted to produce a low affinity 
site by introducing the residue determinants in
that reduce
affinity into equivalent positions of the
subunit. The mutations
P181T and
E182A, singly or combined, do not affect appreciably NmmI affinity (Table I), indicating that substitutions of
other residues unique to the
subunit into the
subunit are required to decrease affinity of NmmI for the 
site.
Residues in NmmI That Interact with Selectivity Determinants in the
and
Subunits--
Because Pro-175 of the
subunit likely
orients Glu-176 to come into close apposition with a cationic residue
on the NmmI toxin, we asked whether an anionic residue at the
homologous position to 176 in the
subunit stabilizes a cationic
residue in the central loop of NmmI. Because Coulombic interactions can
be effective over relatively long distances, and both attractive
(opposite charges) and repulsive (like charges) forces can be
generated, we measured binding of the mutant toxins, K27E, R33E, and
R36E to receptors containing the mutation
E176K. Each pair of
receptor-toxin mutations is equivalent to a charge reversal between
receptor and toxin, and should the distance relationships be
appropriate, charge reversal could preserve a stabilizing interaction,
if Coulombic forces prevail.
Among the cationic residues in NmmI, Lys-27 showed the strongest
interaction with
Glu-176. The receptor mutation
E176K decreases affinity of the 
site for NmmI by nearly 3 orders of magnitude. Similarly, the NmmI mutation K27E decreases affinity of NmmI for the

site by approximately 2.5 orders of magnitude. However, combining both
E176K and K27E results in a complex that is more stable by 4.5 orders of magnitude of NmmI concentration than expected for noninteracting pairs of residues (Fig. 6). Thus charge reversal of
E176K and K27E preserves a stabilizing interaction, suggesting that
electrostatic force between
Glu-176 and Lys-27 is a primary factor
in stabilizing the toxin-receptor complex.
We used thermodynamic mutant cycle analysis to determine free energy of
interaction between charged pairs of residues in the receptor and NmmI
(22) (Scheme I). In this mutant cycle,
the asterisk indicates the presence of a mutation in either
the receptor (R) or the NmmI toxin (T).
The loss of energy, 
G, arising from substitution from
wild type into mutant is calculated from the dissociation constant (KD) as follows.
|
(Eq. 1)
|
The coupling energy, 
GINT, is defined
in terms of the respective dissociation constants (K) of the
complexes,
|
(Eq. 2)
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(Eq. 3)
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(Eq. 4)
|
where the
G° values are the standard free energies
for formation of the toxin/receptor complex. If the mutations do not interact, the two differences in standard free energies should be equal
because the effect of mutating the receptor should be independent of
whether or not toxin is mutated (Equation 3). Similarly, mutating the
toxin should be independent of the receptor mutations (Equation 4). On
the other hand, if the two mutations interact, the bracketed
differences should not be equal. When applied to the
E176K/K27E
pair, mutant cycle analysis reveals a substantial free energy of
interaction of
5.9 kcal/mol (Table I). Similar analysis of the NmmI
mutations, R33E and R36E, reveal modest interaction-free energies of
2.7 and
2.2 kcal/mol, respectively (Table I). The overall results
indicate close approach of cationic residues in the central loop of the
NmmI toxin and
Glu-176 of the binding site, with the most proximal
charged residue being Lys-27 of the toxin.
 |
DISCUSSION |
The
-neurotoxins are a family of three-fingered peptide toxins
found in venom of elapid snakes (7). They have proven to be invaluable
tools for the isolation and study of the nAChR because of their high
affinities and slow rates of dissociation from the receptor (5, 6).
Although the isolated
-subunit of the receptor retains the capacity
to bind
-BgTx, whereas isolated
,
, or
subunits do not,
the
-toxins bind with far lower affinity to the
subunit than to
the intact receptor. Moreover, small agonists and antagonists do not
compete with
-toxin binding to the isolated
subunit at expected
concentrations (23). These observations point to a predominant, but not
sole, contribution to the
-neurotoxin binding coming from the
subunit. Our previous work showed that Val-188, Tyr-190, Pro-197, and
Asp-200 of
subunit contribute to NmmI binding (19). Also
glycosylation at positions 189 and 187, yielding oligosaccharides
uniquely found in cobra and mongoose nAChR, reduced
-BgTx binding
substantially (16).
Although the
subunit appears to be the predominant site of
-toxin binding, chemical cross-linking and mutagenesis studies show
that non-
subunits are close to the site of
-neurotoxin binding
(Refs. 8-14, 16, 17, and see Refs. 3 and 15 for reviews). The results
described here further illustrate the role of neighboring non-
subunits in contributing to high affinity
-toxin binding, as NmmI
binds to 
interfaces of the adult type of nAChR
(
2

) with 3 orders of magnitude lower affinity
than to the 
and 
interfaces of the fetal receptor
(
2

). Binding studies, initially using chimeras
and subsequently point mutants, show that
Thr-176/
Ala-177
(Pro/Glu in
/
) contribute entirely to insensitivity of the 
interface to NmmI. The observation that
-bungarotoxin association is
only slightly affected by the
and
sequence differences suggests
that this region of the
,
, and
subunits is not used
equivalently for stabilization of the entire family of bound
-neurotoxins. At the present time, it is unclear whether
stabilization from this region is unique to some of the short
-neurotoxins, or the long
-neurotoxins, such as
-bungarotoxin,
acquire the bulk of their stabilization energy from other portions of
the structure. Distinct differences in specificity between the short
and long neurotoxins have been noted for the
7 subtype of nAChR
(24).
Residues at the
175/176 positions were previously unrecognized as
determinants of ligand binding. However, they are immediately adjacent
to
Asp-174, which was shown by cross-linking to be ~9 Å away from
Cys-192/193 in the
subunit (25, 26) and was shown to influence the
affinity of quaternary agonists and antagonists (27, 28). Moreover, the
adjacent residues of
Phe-172 (
Asp-178,
Ile-173) are known to
confer site-selectivity to the smaller competitive peptide inhibitors
such as
-conotoxin MI (29) and waglerin (30). The equivalent region
of the
7 subunit (Asp-163, Ile-164, and Ser-165), which presumably
forms a homomeric pentamer of subunits, constitutes part of a putative
Ca2+ binding region that faces the ligand binding site
(31). At the
subunit interface of the binding site, both aromatic
(Tyr-190, Tyr-198) and anionic (Asp-200) residues were mapped to the
-toxin binding surface (19). Here, we identify another anionic
residue in the
subunit, Glu-176, perhaps restricted in its position by a neighboring secondary amino acid Pro-175, on the
subunit, as a
crucial residue for binding.
The significant linkage between Glu-176 and cationic residues in loop
II of the toxin suggests that an electrostatic interaction contributes
to the tight binding of the NmmI/nAChR complex. Because the linkage
obtained from charge reversal is greater for Lys-27 than for Arg-33 and
Arg-36, one would predict that the portion of loop II proximal to loop
III of the toxin, is closest to the
subunit (cf. Fig.
5). In this analysis, the loss of free
energy (
G) associated with a single charge mutation
results from all interactions between the charged residue and its
multiple neighboring residues. The pairwise interactions
(
GINT) resulting in charge reversal of
specified residues in the interacting molecules should then highlight
the strength of interaction coming from the paired charged residues. In
the absence of significant changes in conformation or hydration,

GINT from Equation 2 should largely
reflect the Coulombic interaction between the respective paired
residues (32).

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Fig. 5.
Structure of an
-neurotoxin from Naja mossambica mossambica
(NmmI) and mutations studied. An
energy minimization model of NmmI described in (19), is shown with the
mutated side chains of loop II. The concave face of the toxin is facing
the viewer.
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Extensive studies on a related short neurotoxin, erabutoxin a,
involving mutations at 36 toxin positions clearly revealed the
importance of the tips of loops situated on the concave face of the
toxin (33, 34). These investigations showed that the K27E mutation of
erabutoxin a decreases its affinity more than 100-fold for
Torpedo nAChRs. Photo-activable p-azidobenzoyl
and p-azidosalicyl groups attached to Lys-26 (analogous
position at Lys-27 of NmmI) of neurotoxin II labeled
and
subunits of the receptor upon photolysis (11, 12). Three different
photoactivatable groups attached to the equivalent residue Lys-23 of a
long neurotoxin, toxin 3, also labeled predominantly the
and
subunits in preference to the
subunit (13). Thus mutagenesis and
chemical labeling studies showed a crucial role of lysine at position
27 and its proximity to
and
subunits. Here, our mutant cycle
studies delineate the interaction between Glu-176 of the
subunit
and Lys-27 of NmmI toxin. The largest linkage in loop II between K27E and
E176K (
G =
5.9kcal/mol) and smaller
linkages (
GINT =
1.6 to
3.0
kcal/mol) found previously between K27E and
subunit residues of
Val-188, Tyr-190, Pro-197, and Asp-200 (35) correlate well with the
labeling studies. A more complete
elucidation of
-toxin-receptor interactions should enable us to
orient a docked
-toxin with respect to the subunit interfaces, as
well as refine existing models of the structure of the extracellular
domain of the receptor (4).

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Fig. 6.
Linkage of the free energy of binding between
charge modifications on the ligand and receptor. A and
B, inhibition of the initial rate of
125I-labeled -bungarotoxin binding to cell surface nAChR
expressed as 2  by wild type NmmI (A)
and K27E mutant NmmI (B). C and D,
inhibition of the initial rate of 125I-labeled
-bungarotoxin binding to cell surface nAChR expressed as
2 ( E176K) by wild type (C) and K27E
mutant (D) NmmI -toxin. The dashed line in
D is the predicted curve when the coupling energy
( GINT) between E176K and K27E is 0. The
deviation of observed affinity (D) from that predicted by no
linkage between the residues at the  site (dashed
line) produces a large coupling energy of 5.9 kcal/mol.
|
|
 |
FOOTNOTES |
*
This work was supported in part by United States Public
Health Service Grants GM 18360 (to P. T.) and NS31744 (to S. M. S.).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.
Recipient of a Uehara Memorial Foundation Fellowship.
§
Recipient of a California Tobacco Related Disease Research Program Fellowship.
To whom correspondence should be addressed. Tel.:
619-534-1366; Fax: 619-534-8248.
 |
ABBREVIATIONS |
The abbreviations used are:
nAChR, nicotinic
acetylcholine receptor;
NmmI, Naja mossambica mossambica;
-BgTx,
-bungarotoxin;
wt, wild type;
mt, mutant.
 |
REFERENCES |
-
Unwin, N.
(1993)
J. Mol. Biol.
229,
1101-1124[CrossRef][Medline]
[Order article via Infotrieve]
-
Karlin, A.,
and Akabas, M. H.
(1995)
Neuron
15,
1231-1244[Medline]
[Order article via Infotrieve]
-
Hucho, F.,
Tsetlin, V. I.,
and Machold, J.
(1996)
Eur. J. Biochem.
239,
539-557[Abstract]
-
Tsigelny, I.,
Sugiyama, N.,
Sine, S. M.,
and Taylor, P.
(1997)
Biophys. J.
73,
52-66[Abstract]
-
Chang, C. C.,
and Lee, C. Y.
(1963)
Arch. Int. Pharmacodyn. Ther.
144,
241-257[Medline]
[Order article via Infotrieve]
-
Changeux, J. P.,
Kasai, M.,
and Lee, C. Y.
(1970)
Proc. Natl. Acad. Sci. U. S. A.
67,
1241-1247[Abstract]
-
Endo, T.,
and Tamiya, N.
(1987)
Pharmacol. Ther.
34,
403-451[Medline]
[Order article via Infotrieve]
-
Witzemann, V.,
Muchmore, D.,
and Raftery, M. A.
(1979)
Biochemistry
18,
5511-5518[Medline]
[Order article via Infotrieve]
-
Hamilton, S. L.,
Pratt, D. R.,
and Eaton, D. C.
(1985)
Biochemistry
24,
2210-2219[Medline]
[Order article via Infotrieve]
-
Chatrenet, B.,
Trémeau, O.,
Bontems, F.,
Goeldner, M. P.,
Hirth, C. G.,
and Ménez, A.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
3378-8211[Abstract]
-
Kreienkamp, H. J.,
Utkin, Y. N.,
Weise, C.,
Machold, J.,
Tsetlin, V. I.,
and Hucho, F.
(1992)
Biochemistry
31,
8239-8244[Medline]
[Order article via Infotrieve]
-
Machold, J.,
Weise, C.,
Utkin, Y.,
Tsetlin, V.,
and Hucho, F.
(1995)
Eur. J. Biochem.
234,
427-430[Abstract]
-
Utkin, Y. N.,
Krivoshein, A. V.,
Davydov, V. L.,
Kasheverov, I. E.,
Franke, P.,
Maslennikov, I. V.,
Arseniev, A. S.,
Hucho, F.,
and Tsetlin, V. I.
(1998)
Eur. J. Biochem.
253,
229-235[Abstract]
-
Oswald, R. E.,
and Changeux, J. P.
(1982)
FEBS Lett.
139,
225-229[CrossRef][Medline]
[Order article via Infotrieve]
-
Arias, H. R.
(1997)
Brain Res. Rev.
25,
133-191[CrossRef][Medline]
[Order article via Infotrieve]
-
Kreienkamp, H.-J.,
Sine, S. M.,
Maeda, R. K.,
and Taylor, P.
(1994)
J. Biol. Chem.
269,
8108-8114[Abstract/Free Full Text]
-
Sine, S. M.
(1997)
J. Biol. Chem.
272,
23521-23527[Abstract/Free Full Text]
-
Marchot, P.,
Frachon, P.,
and Bougis, P. E.
(1988)
Eur. J. Biochem.
174,
537-542[Abstract]
-
Ackermann, E. J.,
and Taylor, P.
(1997)
Biochemistry
36,
12836-12844[CrossRef][Medline]
[Order article via Infotrieve]
-
Sine, S. M.,
and Taylor, P.
(1979)
J. Biol. Chem.
254,
3315-3325[Abstract]
-
Sine, S. M.,
and Taylor, P.
(1981)
J. Biol. Chem.
256,
6692-6699[Free Full Text]
-
Horovitz, A.,
and Fersht, A. R.
(1990)
J. Mol. Biol.
214,
613-617[Medline]
[Order article via Infotrieve]
-
Gershoni, J. M.,
Hawrot, E.,
and Lentz, T. L.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
4973-4977[Abstract]
-
Servent, D.,
Winckler-Dietrich, V.,
Hu, H. Y.,
Kessler, P.,
Drevet, P.,
Bertrand, D.,
and Ménez, A.
(1997)
J. Biol. Chem.
272,
24279-24286[Abstract/Free Full Text]
-
Czajkowski, C.,
and Karlin, A.
(1991)
J. Biol. Chem.
266,
22603-22612[Abstract/Free Full Text]
-
Czajkowski, C.,
Kaufmann, C.,
and Karlin, A.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6285-6289[Abstract]
-
Martin, M. D.,
and Karlin, A.
(1997)
Biochemistry
36,
10742-10750[CrossRef][Medline]
[Order article via Infotrieve]
-
Osaka, H.,
Sugiyama, N.,
and Taylor, P.
(1998)
J. Biol. Chem.
273,
12758-12765[Abstract/Free Full Text]
-
Sine, S. M.,
Kreienkamp, H.-J.,
Bren, N.,
Maeda, R.,
and Taylor, P.
(1995)
Neuron
15,
205-211[Medline]
[Order article via Infotrieve]
-
Molles, B. E.,
Kline, E. F.,
Sine, S. M.,
McArdle, J. J.,
and Taylor, P.
(1998)
J. Physiol. (paris)
92,
470[CrossRef] (abstr.)
-
Galzi, J. L.,
Bertrand, S.,
Corringer, P. J.,
Changeux, J. P.,
and Bertrand, D.
(1996)
EMBO J.
15,
5824-5832[Abstract]
-
Faiman, G. A.,
and Horovitz, A.
(1996)
Protein Science
9,
315-316
-
Pillet, L.,
Trémeau, O.,
Ducancel, F.,
Drevet, P.,
Zinn-Justin, S.,
Pinkasfeld, S.,
Boulain, J.-C.,
and Ménez, A.
(1993)
J. Biol. Chem.
268,
909-916[Abstract/Free Full Text]
-
Trémeau, O.,
Lemaire, C.,
Drevet, P.,
Pinkasfeld, S.,
Ducancel, F.,
Boulain, J.-C.,
and Ménez, A.
(1995)
J. Biol. Chem.
270,
9362-9369[Abstract/Free Full Text]
-
Ackermann, E. J.,
Ang, E. T.-H.,
Kanter, R. J.,
Tsigelny, I.,
and Taylor, P
(1998)
J. Biol. Chem.
273,
10958-10964[Abstract/Free Full Text]
-
Prince, R. J.,
and Sine, S. M.
(1996)
J. Biol. Chem.
271,
25770-25777[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.