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Vol. 53, Issue 4, 787-794, April 1998
-Conotoxin M1 Binding
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093 (N.S., P.M., C.K., H.O., B.M., P.T.) and Department of Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55455 (S.M.S.)
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Summary |
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The two binding sites in the pentameric nicotinic acetylcholine
receptor of subunit composition
2

are formed by
nonequivalent
-
and
-
subunit interfaces, which produce
site selectivity in the binding of agonists and antagonists. We show by
sedimentation analysis that 125I-
-conotoxin M1 binds
with high affinity to the
-
subunit dimers, but not to
-
dimers, nor to
,
, and
monomers, a finding consistent with
-conotoxin M1 selectivity for the 
interface in the intact
receptor measured by competition against
-bungarotoxin binding. We
also extend previous identification of
-conotoxin M1 determinants in
the
and
subunits to the
subunit interface by mutagenesis of
conserved residues in the
subunit. Most mutations of the
subunit affect affinity similarly at the two sites, but Tyr93Phe,
Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a
site-selective manner. Mutant cycle analysis reveals only weak or no
interactions between mutant
and non-
subunits, indicating that
side chains of the
subunit do not interact with those of the
or
subunits in stabilizing
-conotoxin M1. The overall findings
suggest different binding configurations of
-conotoxin M1 at the
-
and
-
binding interfaces.
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Introduction |
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Nicotinic
acetylcholine receptors are pentamers of homologous subunits with
composition
2

that form a ring around
a central channel (Galzi and Changeux, 1994
; Karlin and Akabas, 1995
).
Two of its five subunit interfaces, 
and 
, form binding
sites for the neurotransmitter acetylcholine. These two binding
interfaces are not identical in their affinities for agonists and
competitive antagonists (Damle and Karlin, 1978
; Neubig and Cohen,
1979
; Weiland and Taylor, 1979
; Sine and Taylor, 1981
). Because the
subunit is common to each binding interface, differences in affinity
are attributed to the contributions of the
and
subunits (Blount and Merlie, 1989
; Petersen and Cohen, 1990
, Sine and Claudio, 1991
).
Recent studies showed that certain
-conotoxins, 12-14-amino acid
disulfide-linked peptides isolated from venom of cone snails (Myers
et al., 1991
, 1993
), bind with unusual selectivity to one of
the two ligand binding sites on mouse and Torpedo
californica receptors (Kreienkamp et al.,
1994
; Hann et al., 1994
; Utkin et al., 1994
;
Groebe et al., 1995
; Sine et al., 1995a
).
-Conotoxin M1 binds with high affinity to the 
site of the
mouse receptor (KD = 0.5 nM), whereas it binds five orders of magnitude
less tightly to the 
site
(KD = 20 mM) (Kreienkamp et al., 1994
; Sine
et al., 1995a
).
-Conotoxin Ml is unique in that its
degree of selectivity is greater than for any known ligand, and its
site preference is opposite to that of curariform antagonists, which bind more tightly to the 
site (Blount and Merlie, 1989
; Petersen and Cohen, 1990
; Sine and Claudio, 1991
).
The high degree of sequence identity between the
and
subunits
suggests that the polypeptide chains of the two subunits fold into
similar basic scaffolds. Thus residues in equivalent positions of the
linear sequence are predicted to occupy similar positions in three
dimensional space. This idea is supported by the striking finding that
three residues in equivalent positions of the
and
subunits
confer virtually all of the selectivity for
-conotoxin Ml (Sine
et al., 1995a
). Residues in the
subunit that stabilize
-conotoxin Ml have not been identified, but mutagenesis studies with
agonists and antagonists revealed three regions of the
subunit that
contribute to the binding interface (Sine et al., 1994
;
Galzi et al., 1991
; Middleton and Cohen, 1991
; Tomaselli et al., 1991
; O'Leary and White, 1994
; O'Leary et
al., 1994
; Sugiyama et al., 1996
). These three regions
differ from binding site regions in the
and
subunits, because
they are predicted to be located on the opposite face of the subunit.
In this study we employ radioiodinated
-conotoxin M1 to show
directly that its binding requires an intact subunit interface; high
affinity binding is found only at the 
interface. We then examine
through residue replacement the relationships between amino acid
determinants in the
subunit and those in the
and
subunits
that govern
-conotoxin M1 binding.
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Materials and Methods |
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Radioiodination of conotoxin M1.
-Conotoxin M1 (American
Peptide Co.), 0.75 nmol (1.125 mg), was iodinated at its single
tyrosine (Tyr-12) with 0.1 mCi of Na125I
(Amersham) and 2.5 mg of lactoperoxidase (Sigma) in 100 ml of a 50 mM NaPO4 buffer, pH 7.5. Free iodide
was removed by selective adsorption on a Dowex 1X-8 (Bio-Rad) cationic
resin (Marchot et al., 1993
). Labeled
-conotoxin M1 was
stored as a 0.5 mM solution in a 1:1 methanol:50
mM NaPO4 buffer, pH 7.5, at
20°.
Dilute solutions (1 mM) were prepared in 1 mg/ml bovine
serum albumin, 50 mM NaPO4, pH 7.5, stored at 4°, and used within the next 3 weeks. Specific activities
of 100 Ci/mmol were achieved, which corresponded to 0.05 atom of iodine
incorporated per molecule of
-conotoxin M1. The ratio of labeled
species was kept low to minimize formation of diiodo-
-conotoxin M1.
Cell transfections.
Human embryonic kidney 293 cells were
transfected with cytomegalovirus-based expression vectors containing
the respective cDNAs encoding the individual subunits by
Ca3(PO4)2
precipitation (Sine, 1993
; Kreienkamp et al., 1994
).
Typically plasmids containing cDNAs encoding
,
,
, and
subunits in the weight ratio 2:1:1:1, and
,
, and
, or
,
, and
in the ratio of 2:1:2 were transfected. The transfections
of cDNAs encoding the above four and three subunits yielded pentameric
receptors
2

,
2
2 or
2
2 expressed at the cell surface (Sine and Claudio, 1991
), whereas transfection of a
cDNA encoding
or
subunit and cotransfection of two cDNAs encoding
and
or
and
required permeabilization of the
cells to detect monomeric, dimeric, or tetrameric combinations of
subunits within the cells (Green and Claudio, 1993
; Kreienkamp et
al., 1995
).
Association of 125I-
-conotoxin M1 and
125I-
-bungarotoxin with isolated and assembled
subunits.
Three days after transfection, cells were harvested by
gentle agitation in phosphate-buffered saline, pH 7.4, containing 5 mM EDTA. After low speed sedimentation, cells were
permeabilized with saponin-containing buffer (10 mM, EDTA,
0.1% bovine serum albumin, and 0.5% saponin in 10 mM
NaPO4, pH 7.4), and then incubated on ice with 5 nM 125I-
-bungarotoxin (specific
activity of 8-16 mCi/mg; DuPont-New England Nuclear, Boston, MA) or
with 125I-
-conotoxin M1 at the specified
concentrations. Cells were then sedimented and washed free of excess
unbound ligand with potassium Ringer's buffer. The pellets were
solubilized on ice in 1.0% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris·HCl, pH 7.5. After 4 hr,
supernatants were layered over 3-30% sucrose gradients containing the
same detergent buffer. Layered gradients were centrifuged in a Beckman
SW41 rotor at 40,000 rpm for 22 hr at 4°. Fractions were collected
and assayed; the S values were determined as previously described (Kreienkamp et al., 1995
; Sugiyama et
al., 1996
).
-Conotoxin competition with
125I-
-Bungarotoxin.
Harvested cells were
resuspended in potassium Ringer's buffer to measure ligand binding to
receptors expressed on the cell surface. Specified concentrations of
-conotoxin M1 were added to each aliquot of cell suspension 60 min
before measurement of the initial rate of
125I-
-bungarotoxin binding. The fractional
reduction in the initial rate corresponds to the fractional occupation
of sites by
-conotoxin M1 (Sine and Taylor, 1981
; Sine et
al., 1995a
). KD values for each
mutant are given as averages from two separate cell transfections. Sufficient cells were transfected to generate an entire concentration profile with duplicate samples measured at each concentration. The
KDvalues typically varied by less
than 20%.
Site-directed mutagenesis.
Mutations of the individual
subunits were generated by the method of Kunkel (1985)
; the entire
mutagenic insert was sequenced to verify the mutation and rule out
random polymerase errors.
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Results |
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125I-
-Conotoxin M1 binding to the receptor
subunits.
When cDNA encoding
subunit is cotransfected with
cDNAs encoding either the
or
subunit, the resulting assembled
subunits are retained within the cell. To detect association of labeled
-conotoxin, with monomeric subunits and the 
and 
assembled oligomers, the cells were permeabilized with saponin,
incubated with labeled toxin, washed, solubilized, and centrifuged into sucrose density gradients. The gradients resolve free toxin as well as
labeled toxin associated with monomeric, dimeric, and tetrameric
subunit oligomers (Fig. 1).
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subunit, the
and
pairs or the
and
pairs resulted in 125I-
-bungarotoxin
association with
subunit, the assembled 
and 
subunit
dimers, and the 


tetramer (Fig. 1, B1-B3). In contrast,
association of 125I-
-conotoxin M1 is detected
only with the 
dimer, but neither with the 
dimer nor with
the 


tetramer (Fig. 1, C1-C4). The absence of a peak or
shoulder corresponding to
subunit monomer when cells were
cotransfected with either
and
or
and
pairs of cDNAs
indicates that free subunit monomer does not bind
125I-
-conotoxin M1 with high affinity. To rule
out the possibility that the
subunit rather than the 
dimer
is responsible for
-conotoxin binding, we examined
125I-
-conotoxin M1 association with the
isolated
subunit where expression of
subunit after cDNA
transfection has been confirmed with Western blot analysis using a
subunit specific monoclonal antibody (mAb166) (Keller S and Taylor P,
unpublished observations). As shown in Fig. 1, C2, only a free toxin
peak is observed indicating that
125I-
-conotoxin M1 does not associate with the
subunit monomer.
Selectivity of 20 nM
125I-
-conotoxin M1 for the 
interface is
revealed by the lack of dissociation of the complex as it migrates into
the gradients. The results are consistent with previous studies of
-conotoxin M1 competition with
125I-
-bungarotoxin binding to the intact
receptor on the cell surface and cell surface receptors devoid of
either the
or
subunit (Kreienkamp et al., 1994
-conotoxin M1, binding to the 
subunit dimer could not be detected above background levels (Fig. 1,
C5), yet parallel plates of cells expressing the 
subunit
combination showed 125I-
-bungarotoxin
association with 
dimer and 


tetramer (Fig. 1, B3).
Identification of
subunit determinants for
-conotoxin M1
binding.
Site-directed labeling and mutagenesis studies have
defined three linearly distinct regions in the
subunit (regions
A-C) that contribute to the ligand binding site (Dennis et
al., 1988
; Galzi et al., 1991
; Middleton and Cohen,
1991
; Tomaselli et al., 1991
; O'Leary and White, 1992
;
O'Leary et al., 1994
; Sine et al., 1994
; Fu and
Sine, 1994
; Keller et al., 1995
). Each region, presumably existing as a loop at the subunit interface, contains conserved aromatic residues, including Y93, W149, Y190, and Yl98 (Fig.
2). Here we mutate residues in these
three regions and measure changes in
-conotoxin M1 affinity. Most
mutations are substitutions for the aromatic residues, but also include
substitutions of residues differing between muscle and neuronal
subunits. Binding of
-conotoxin M1 was measured by competition
against the initial rate of 125I-
-bungarotoxin
binding, as described previously (Sine and Taylor, 1981
).
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Mutation of conserved tyrosines in regions A, B, and C.
Mutation of Y93, Y190, and Y198 to aliphatic hydroxyl side chains (S or
T) reduces affinity for
-conotoxin M1 (Fig.
3 and Table
1). Furthermore, the Y190T and Y198T
mutations result in a selective influence for the affinity of the

site is affected to much greater extent than the 
site.
Removal of the aromatic hydroxyl at Y151, Y190, and Y198 has no effect
on
-conotoxin M1 affinity, whereas the Y93F mutation enhances
affinity in a site-selective manner, increasing affinity for the 
site without affecting the 
site (Fig.
4 and Table 1). Thus, mutation of the
four conserved tyrosines affects
-conotoxin affinity in a manner
similar to other antagonists (O'Leary et al., 1994
; Sine et al., 1994
). Aliphatic hydroxyl substitutions dramatically
reduce affinity for
-conotoxin as observed previously for agonists
and antagonists, whereas the removal of the hydroxyl group by
substitution of phenylalanine either has little influence or enhances
affinity (Sine et al., 1994
, 1995b
; Tomaselli et
al., 1991
; O'Leary et al., 1994
). Interestingly, Y93F
increases affinity of
-conotoxin (Fig. 4), whereas Y198F enhances
d-tubocurarine affinity (Fu and Sine, 1994
; Sine et
al., 1994
).
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Mutation of charged, aromatic, and proline residues in region
C.
-Conotoxin M1 is amidated on its carboxyl-terminus, lacks
negatively charged side chains, but contains three or four positively charged side chains depending on pH. We therefore focused on charged residues in loop C, which differ between muscle and neuronal
subunits (Fig. 2). Replacement of positively charged residues with
R182V or K185E, or insertion of a positive charge with F189K, fails to
affect
-conotoxin M1 affinity (Table 1). By contrast, inserting a
positive charge with V188K selectively decreases
-conotoxin Ml
affinity for the 
site without affecting affinity for the 
site (Fig. 4 and Table 1). Thus, electrostatic interactions with
-conotoxin M1 seem localized to position 188 and specifically affect
the 
site.
-conotoxin M1
affinity (Table 1).
A small loop bounded by prolines at the 194 and 197 positions is a
characteristic feature of the
1 subunit, not found in neuronal
subunits (Fig. 2). Deletion of P194 or the P194L substitution has
little effect on
-conotoxin M1 binding. The P197I mutation reduces
-conotoxin M1 affinity 2-4-fold at both sites, whereas the T195E
mutation also reduces
-conotoxin M1 affinity, primarily at the

site.
Mutations in loop B.
Mutations of the conserved W149 and Y151
to phenylalanine do not significantly affect
-conotoxin Ml binding
(Table 1). The lack of effect of W149F on
-conotoxin M1 affinity
contrasts with the large reduction in agonist affinity associated with
this mutation (Sine et al., 1994
). Previous studies showed
that D152N introduces a glycosylation consensus site that becomes
glycosylated, and further that agonist and antagonist affinities are
markedly reduced (Sugiyama et al., 1996
). For
-conotoxin
M1, D152N reduces affinity slightly for the 
site without
affecting the 
site. Thus, the aromatic residues examined in
region B make no contribution to
-conotoxin M1 affinity, whereas the
negative charge at position 152 enhances its affinity.
Mutagenesis of residues in both the
subunits and non-
subunits.
Previous studies identified three residues in equivalent
positions of the
and
subunits that confer higher affinity of
-conotoxin M1 for the 
over the 
interface (Sine
et al., 1995a
). To account for the selective effect of
Y93F and
V188K for the 
interface, we reasoned that either
-conotoxin M1 affinity is enhanced by interaction between
determinants in the
and
subunits or
-conotoxin M1 orients
differently at the 
and 
interfaces. Thus we investigated
these two possibilities by examining
-conotoxin Ml binding to
receptors containing mutations in both the
and
subunits.
Y93F and
V188K
incorporated into pentameric receptors lacking the
subunit. The
resulting cell surface receptors have the composition
2
2, which should
contain two equivalent 
binding sites (Sine and Claudio, 1991
Y93F into these pentamers increases
-conotoxin M1
affinity, whereas incorporating
V188K decreases affinity for both

sites (Fig. 5A and Table
2), as observed for one of the two sites
in
2

pentamers (Fig. 4 and Table 1).
Moreover, when coexpressed with the series of mutant
subunits, the
Y93F and
V188K mutations affect
-conotoxin M1 affinity to the
same extent as observed when coexpressed with the wild-type
subunit
(Fig. 5A). Thus,
Y93F and
V188K do not interact with residues in
the
subunit that confer selective binding of
-conotoxin M1.
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Y93F nor
V188K significantly affect affinity
when paired with the wild-type
subunit and expressed as
2
2. However, they
affect affinity strongly when paired with the triple mutant of the
subunit (
SYI) that increases affinity to approach that conferred by
the
subunit (Fig. 5). These findings suggest that other residues
unique to the
subunit nullify the effects of
Y93F and
V188K,
perhaps by conferring a different orientation of
-conotoxin M1 at
the 
and 
interfaces. Alternatively, constraints imposed by
the
subunit may preclude Y93F in the
subunit from achieving a
higher affinity than in the wild-type receptor
-
interface.
We also examined the influence of the a Y93F and V188K mutations on
-conotoxin M1 affinity when the receptor is assembled from four
distinct subunits as
2

, rather than
as
2
2 or
2
2.
-Conotoxin
M1 affinity for the 
site is slightly higher, whereas the
affinity for the 
site is slightly lower in the
2

pentamer, compared with the
pentameric combinations of the three respective subunits (Table 2 and
Fig. 5B). Hence, the difference in
KDvalues for the two sites is larger
in the receptor assembled from four distinct subunits
(
2

) than three
(
2
2 or
2
2). This may
reflect longer range interactions between binding sites. Nevertheless,
the influence of the
subunit mutations is the same in the
pentameric assemblies of four and three distinct subunits (Fig. 5B).
The
Y190T and
Y198T mutations show a predominant, but not
completely selective, influence on reducing the affinity of the high
affinity, 
interface (Fig. 3A and Table 1). When these mutations
are coexpressed as pentamers with the
subunit triple mutant
(
SYI), a reduction in
-conotoxin M1 affinity similar to that
found for the
subunit is seen; the absolute affinities are enhanced
over coexpression with wild-type
because of the
SYI mutation
(Fig. 6). Examination for the opposite
situation, the substitution of
subunit containing side chains into
the
template, yielded diminished receptor expression with the
mutant
subunits and precluded a precise quantitation of affinity.
Nevertheless, the influence of
Y190T and
Y198T mutations seems to
depend on the binding affinity of
-conotoxin M1 and, here again, may
reflect a slightly different binding position for
-conotoxin M1
between the high and low affinity binding sites.
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Discussion |
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Subunit contributions to
-conotoxin M1 association.
Radioiodination to form 125I-
-conotoxin M1
enables one to monitor directly the
-conotoxin association with
various subunit combinations. High affinity binding where bound
125I-
-conotoxin M1 is retained by the receptor
subunits after sedimentation is evident only for the 
dimer. The
same procedure shows
-bungarotoxin association with
subunit
monomers, 
dimers, 
dimers, and 


tetramers.
These findings not only document directly the site selectivity of
-conotoxin M1, but also reveal that
-conotoxin M1 requires an
intact 
subunit interface for high affinity binding.
Residues conferring
-conotoxin M1 selectivity.
Previous studies have defined three residues in the
subunit (S36,
Y113, and I178), which confer high affinity binding of
-conotoxin M1
to the 
interface (Sine et al., 1995a
). When substituted with the corresponding residues in the
subunit (K34, S111, and F172), this triad of mutations in the
template confers an
-conotoxin M1 affinity approaching that of the
subunit (Sine et al., 1995a
). To account for 
and 
interfaces
showing nearly a 10,000-fold difference in
-conotoxin M1 affinity,
either the
subunit diminishes the influence of determinants on the
subunit to
-conotoxin binding or determinants on the
subunit
are major contributors to the high affinity of
-conotoxin M1. In
turn, the
subunit could enhance affinity either directly through
its own interactions with conotoxin or by altering the conformation of
the
subunit. Because the differences in
-conotoxin M1 affinity between 
and 
interfaces are diminished and actually
inverted for the T. californica receptor (Hann et
al., 1994
; Utkin et al., 1994
; Groebe et
al., 1995
; Sine et al., 1995a
) and affinity of the

subunit interface is intermediate to the 
and 
interfaces in mouse (Sine S, unpublished observations), the
,
,
and
subunits likely contribute to stabilization of the
-conotoxin complex both directly and by affecting the conformations
of the neighboring
subunit.
subunit known to
influence agonist and alkaloid antagonist binding to the receptor.
Because several of the
-conotoxins such as M1 are relatively
selective for the muscle subtype of receptor (i.e.,
1
subunit-containing receptors), we also relied on differences in the
sequence between
1 and the neuronal
subunits (
2,
3,
4,
and
7) of receptor. In the
1 subunit, the region between residues
180 and 200 has been particularly well studied. Two tyrosines, Y190 and
Y198, are conserved in all
subunits, and their modification affects
the binding of both agonists and antagonists (Tomaselli et
al., 1991
-conotoxin M1 association. However,
Y93F enhances
-conotoxin affinity. Removal of aromaticity by
substituting T for Y has a substantial influence on both
-conotoxin
M1 (Table 1 and Figs. 3 and 6), and the non-peptide antagonists (Sine
et al., 1994
interface. Thus, aromaticity may be required to stabilize the quaternary amine moiety as well as the cationic peptide.
Several further modifications of residues in this region showed that
creating a positive charge at residue 188 markedly decreased binding
affinity, whereas a negative charge at this position slightly enhanced
affinity; these changes were evident only at the 
interface. Modification of residues between the two prolines at position 197 produced complex behavior: substitutions or deletions at position 194 were largely without influence, whereas substitutions at position 197 slightly lowered
-conotoxin M1 affinity. The T195E mutation reduced
-conotoxin M1 affinity selectively at the 
interface. Unfortunately, we were unable to obtain reproducible binding with the
T196E mutation. The cumulative influence of residues 194-197, when
modified to residues found in neuronal receptors (Fig. 2), could
account for part of the selectivity of
-conotoxin M1 for the muscle
type of receptor.
The Y93F mutation enhances
-conotoxin M1 binding affinity at the low
affinity, 
site, whereas substitution of serine decreases the
affinity at this site (Fig. 4). Modifications of aromatic residues at
positions 149 and 151 are without influence, whereas the D152N mutation
selectively reduces the affinity at the 
interface (Fig. 3).
Hence, the three regions of linear sequence known to affect the
affinity of agonists and nonpeptidic antagonists also influence
-conotoxin binding. However, the similarities of residue
contributions to
-conotoxin M1 and other nonpeptidic antagonists
apply to regional segments of sequence but not necessarily to the
individual amino acids.
Relationship between
subunit mutations and
/
subunit mutations.
We examined whether linkage relationships exist
between mutations in the
subunit and those in
and
subunits
or whether the determinants on each subunit act independently of each
other as would be expected if they bound to different portions of the
-conotoxin molecule. A strong linkage relationship is apparent between S36 and I178 on the
subunit, where the individual
substitutions, S36K or I178F, has a small or no influence on
-conotoxin KD values, yet when
both substitutions are made, a marked loss in affinity or increase in
KD evident (Sine et al.,
1995a
). Using a mutant cycle analysis (Carter et al., 1984
)
for mutations at corresponding positions 36 and 178 in the
subunit
template, we may set the following cycle:
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|
|
-conotoxin and the receptor site are subscripted by the residues at
positions 36 and 178. S1,
S2, I1, and
I2 designate the ratio of dissociation
constants for the species at the base of the arrow relative to the tip
of the arrow. The values are represented as absolute values without
reference to sign.
Hence, a comparatively large linkage or coupling energy is seen between
positions 36 and 178 in the
subunit for the binding of
-conotoxin M1. When similar mutant cycles were constructed to relate
mutations in the
subunit with those in
and
subunits, linkage relationships between the three
/
positions at residues 34/36, 111/113, and 172/178 with residues 93 and 188 in the
subunit
were not evident (
0.35 kcal/mol);
rather, the energetic contributions to
-conotoxin M1 binding of
residues in the
subunit seem independent of those in the
and
subunits (compare Fig. 5). Hence, the coupling energy contributing
to
-conotoxin binding occurs within rather than between subunits.
The independence of mutations between subunits indicate that distinct
surfaces of the
-conotoxin molecule interact with the
and
/
subunit faces.
Our findings that the 
and 
interfaces possess disparate
affinities for
-conotoxin M1 and that certain residues in the
subunit selectively affect binding at the
or
interface, suggest
that the amino acid contributions from the common
subunit as well
as the distinct
and
subunits differ in the stabilization of
-conotoxin M1. Hence, the orientations of the bound
-conotoxin molecules at the two sites are likely to be distinct.
Recent x-ray crystallographic (Guddat et al., 1996
-conotoxin-G1, P1VA, and Pn1A show a triangular, wedgelike
structure stabilized by the disulfide bonds between Cys3 and Cys8 and
between Cys4 and Cys14. Fitting the sequence of
-conotoxin M1 into
the structural template shows that the amino terminus (either Gly1 or
the side chain of Arg2), Pro6, and Arg10 are found at the vertices of
the triangle. The arginines at positions 2 and 10 are some 15 Å apart, suggesting that different receptor subunits could harbor the anionic sites that stabilize the cationic loci. Because, in addition to the
amino terminus, only one cationic charge at position 10 is conserved as
Arg or Lys among the
-conotoxins (Myers et al., 1991
-conotoxins and analysis of their
binding to mutant receptors should yield further details on the
orientation of the bound peptide.
| |
Acknowledgments |
|---|
We thank Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA) for the generous gift of MAb166.
| |
Footnotes |
|---|
Received December 1, 1997; Accepted January 12, 1998
1 Current affiliation: Yokohama City University, Yokohama, Japan.
2 Current affiliation: Centre National de la Recherche Scientifique UAR 6560, Institut Federatif de Recherche Jean Roche, Laboratoire de Biochemie, F-13916 Marseille Cedex 20, France.
This work was supported by United States Public Health Service Grants GM18360 (P.T.) and NS31744 (S.M.S.).
Send reprint requests to: Dr. Palmer Taylor, Dept. of Pharmacology, Basic Science Bldg./0636, University of California-San Diego, 9500 Gilman Dr., La Jolla, CA 93093-0636.
| |
Abbreviations |
|---|
HEK, human embryonic kidney.
| |
References |
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|
|
|---|
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subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions.
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