Subunit Interface Selectivity of the alpha -Neurotoxins for the Nicotinic Acetylcholine Receptor*

Hitoshi OsakaDagger , Siobhan Malany§, Joan R. Kanter, Steven M. Sine, and Palmer Taylorparallel

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha -neurotoxins, binds with high affinity to alpha gamma and alpha delta subunit interfaces (KD~100 pM) but binds with markedly reduced affinity to the alpha epsilon interface (KD~100 nM). By constructing chimeras composed of portions of the gamma  and epsilon  subunits and coexpressing them with wild type alpha , beta , and delta  subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gamma Pro-175 and gamma Glu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon , confer low affinity. To identify an interaction between gamma 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 gamma 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 gamma  subunit surface of the binding site interface.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nicotinic acetylcholine receptor (nAChR)1 found in muscle is a pentamer composed of four homologous subunits present in the stoichiometry alpha 2beta gamma delta (fetal subtype) or alpha 2beta epsilon delta (adult subtype). The subunits are arranged in a circular manner to surround a central channel in the order, alpha gamma alpha delta beta or alpha epsilon alpha delta beta (1-3). The two binding sites for agonists, competitive antagonists, and the slowly dissociating alpha -neurotoxins are formed at interfaces between the alpha delta and alpha gamma (epsilon ) 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 alpha  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-alpha subunits, appearing on the opposite face of the subunit, contain major determinants for ligand selectivity; in the gamma  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, alpha -bungarotoxin (5), and the use of labeled alpha -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 alpha -neurotoxin family, which show a common basic structure consisting of three polypeptide loops emerging from a small globular core (7). alpha -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 (alpha epsilon or alpha gamma and alpha delta ), 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 alpha -neurotoxin within the nAChR binding sites have been examined by cross-linking chemically modified (8, 9) or photoactivatable derivatives of alpha -neurotoxin (10-13) and by simple ultraviolet irradiation without chemical modification (14). These labeling studies have suggested contacts with both alpha  and non-alpha 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 alpha  (16) and non-alpha subunits (17) that contribute to alpha -toxin binding. Although most alpha -toxins do not distinguish between the two sites on the receptor, an alpha -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 alpha -toxins and the non-alpha subunits at the binding site. Previous work showed that the alpha gamma and alpha delta 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 alpha  subunit common to both sites, resulted in nonequivalent reductions in affinity at the alpha gamma and alpha delta binding sites (19). Here we examine binding of recombinant NmmI alpha -toxin to fetal and adult mouse AChRs and find that the affinity of NmmI for the alpha epsilon interface is 3 orders of magnitude lower than for the alpha gamma and alpha delta interfaces. Using subunit chimeras and site-directed mutations in gamma  and epsilon  subunits, we show that the enhanced affinity conferred by the gamma  over the epsilon  subunit arises from Pro-175 and Glu-176 in the gamma  subunit. Mutant cycle analysis shows that Glu-176 interacts with cationic residues in loop II of the NmmI alpha -toxin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- alpha -Conotoxin MI was purchased from American Peptide Company. 125I-labeled alpha -bungarotoxin (alpha -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 alpha -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: alpha  (15 µg)/beta (7.5 µg)/gamma or epsilon  (7.5 µg)/delta (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 alpha -bungarotoxin. Dissociation constants of the ligands were determined from their fractional reduction of the initial rate of 125I-labeled alpha -bungarotoxin association (20, 21). Appropriate concentrations of alpha -conotoxin MI were used to block the alpha delta interface but not that of alpha gamma , when the distinctions in affinity for the alpha gamma and alpha delta interfaces were not obvious (19).

Rate Measurements-- The association rate for 125I-labeled alpha -bungarotoxin was measured using a alpha -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insensitivity of the alpha epsilon Site to NmmI-- Certain competitive antagonists distinguish between the two binding sites of the nAChR because of species or subtype differences in the non-alpha subunits that form the alpha gamma , alpha delta , and alpha epsilon binding sites. We therefore compared binding of NmmI to mouse fetal (gamma  containing) and adult (epsilon  containing) nAChRs expressed in HEK 293 cells. As described previously, NmmI does not distinguish between alpha gamma and alpha delta 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 alpha delta and alpha epsilon sites of the adult receptor, binding to the alpha epsilon site with three orders of magnitude lower affinity (KD = 130 nM; Fig. 1). Thus the epsilon  subunit of the adult receptor contains residues that confer insensitivity to NmmI.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   Sensitivity of nAChR expressed as alpha 2beta gamma delta and alpha 2beta epsilon delta in HEK cells to alpha -neurotoxin from Naja mossambica mossambica (NmmI). HEK cells were transfected with cDNAs encoding wild type subunits to form cell surface receptors with the composition alpha 2beta gamma delta () and alpha 2beta epsilon delta (open circle ). alpha -Neurotoxin (NmmI) binding was measured by competition with an initial rate of alpha -bungarotoxin binding. kobs/kmax is the ratio of initial rates for 125I-labeled alpha -bungarotoxin binding in the presence and absence of NmmI. Data are plotted according to the equation: kobs = 0.5 kT,alpha delta ([NmmI]/([NmmI] + Kalpha delta )) + 0.5 KT,alpha epsilon ([NmmI]/([NmmI] + Kalpha epsilon )), where kT,alpha delta and kT,alpha epsilon are the alpha -bungarotoxin association rates for the alpha delta and alpha epsilon sites, Kalpha delta and Kalpha epsilon are the equilibrium dissociation constant for NmmI of the respective sites, and kmax = 0.5 kT,alpha delta  + 0.5 kT,alpha epsilon . For alpha 2beta gamma delta , the data are fit to a single class of sites. The fraction of sites with an affinity corresponding to alpha delta and alpha epsilon is reflected by the inflection. The apparent fraction of sites of alpha delta (0.62) to alpha epsilon (0.38) in alpha 2beta epsilon delta that is greater than the predicted equal population of sites since kT,alpha delta  > kT,alpha epsilon (cf. Fig. 2).

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 alpha delta and alpha epsilon sites. This apparent inequality could arise from distinct rates of association of the reporter ligand 125I-labeled alpha -bungarotoxin with alpha delta and alpha epsilon sites. To examine this possibility, we compared time courses of association of 125I-labeled alpha -bungarotoxin for fetal and adult nAChRs (Fig. 2). For the alpha 2beta gamma delta 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 alpha delta and alpha gamma sites. On the other hand, for the alpha 2beta epsilon delta 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 alpha -bungarotoxin binding (17). The apparent fraction of the two sites will be biased toward the site with the more rapid rate of alpha -bungarotoxin binding.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Kinetics of 125I-labeled alpha -bungarotoxin association with the nAChR expressed as alpha 2beta gamma delta and alpha 2beta epsilon delta in HEK cells. Top panel, association of 5 nM 125I-labeled alpha -bungarotoxin with 200 pM wild type alpha 2beta gamma delta () and alpha 2beta epsilon delta (open circle ) receptors. The data for alpha 2beta gamma delta are fit by a single exponential approach to equilibrium with a kon of 3.6 ± 0.7 × 106 M-1 min-1, whereas for alpha 2beta epsilon delta 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 alpha -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 alpha 2beta gamma delta and 2.6 ± 0.6 × 10-4min-1 for alpha 2beta epsilon delta .

We also compared time courses of dissociation of 125I-labeled alpha -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 alpha 2beta gamma delta and koff = 2.6 ± 0.6 × 10-4 min-1 for alpha 2beta epsilon delta (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 alpha -bungarotoxin of 69 pM (alpha delta and alpha gamma ) for alpha 2beta gamma delta and 67 pM (alpha delta ), 260 pM (alpha epsilon ). These kinetic experiments demonstrate that alpha -bungarotoxin, has far less capacity than NmmI to distinguish between the alpha gamma and alpha epsilon sites.

Molecular Basis of Insensitivity of the alpha epsilon Site for NmmI Toxin-- The gamma  and epsilon  subunits show high sequence identity in the extracellular domains (54% in mouse), and homologous residues should have virtually identical locations for their alpha -carbon backbone positions. Yet, NmmI binds 1000-fold more tightly to the alpha gamma than to the alpha epsilon site. To determine the structural basis of NmmI selectivity, we constructed subunit chimeras containing portions of the gamma  subunit substituted into the epsilon  subunit. Each chimera was coexpressed with complementary alpha , beta , and delta  subunits, followed by measurements of NmmI binding. We first screened with chimeras containing gamma  sequence from the amino terminus to junctions ranging from positions 74 to 173 of the epsilon  subunit. Each of these chimeras confers low affinity for NmmI, characteristic of the wild type alpha epsilon 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 alpha gamma site (KD = ~80 pM; Fig. 3). Thus residue differences between positions 174 and 177 confer NmmI selectivity for the alpha gamma over the alpha epsilon site.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   NmmI alpha -toxin association with the nAChR formed by transfection of cDNAs encoding chimeras of the gamma  and epsilon  subunits, along with alpha , beta , and delta  subunits. Left, schematic representation of chimeric cDNAs formed between the gamma  and epsilon  subunits. (gamma 74epsilon ) denotes that the amino-terminal 74 amino acids are constructed from gamma  subunit (shaded), and residues carboxyl-terminal to this position come from epsilon  subunits (nonshaded). M1-4 denotes the putative transmembrane regions. Right: The dashed vertical line represents the KD for NmmI binding to the wild type alpha gamma interface. The dashed bars show the actual KD for each chimera (top ruler) and the log [KD,mt/KD,wt ] (bottom ruler). Delta Delta G values can be obtained from multiplying by 2.3 RT.

                              
View this table:
[in this window]
[in a new window]
 
Table I
The influence of alpha -toxin and receptor mutations at the gamma , delta , and varepsilon  subunits on Naja mossambica mossambica (NmmI) alpha -toxin association with the nicotinic acetylcholine receptor
Dissociation constants were calculated from competition with the initial rate of the 125I-labeled alpha -bungarotoxin binding. Receptor was expressed as alpha 2beta gamma delta or alpha 2beta varepsilon delta by transfection of cDNAs encoding four respective sets of subunits. KD is dissociation constant for alpha delta , alpha gamma , or alpha varepsilon 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. Delta Delta G is free energy of binding calculated from Equation 1 in the text. Delta Delta GINT was calculated using Equation 2 in the text. Values less than unity were inverted and indicated with a minus sign.

Sequence comparison of the gamma  and epsilon  subunits reveals only two mismatched residues between positions 174 and 177 (Fig. 4A). We therefore constructed point mutations at these two positions of the gamma  subunit and measured NmmI binding to the resulting mutant receptors. The point mutations gamma P175T and gamma E176A reduce affinity to values intermediate to those of the wild type alpha gamma and alpha epsilon sites (P175T, KD,mt/KD,wt = 36; E176A, KD,mt/KD,wt = 16; Fig. 4, Table I). Combining the two mutations into a single gamma  subunit reduces affinity to approach that of the wild type alpha epsilon site (Fig. 4, Table I). The converse double mutation in the epsilon  subunit, epsilon T176P and epsilon A177E, increases NmmI affinity to equal that of the wild type alpha gamma site (Fig. 4, Table I). Thus, the residue pairs at homologous positions gamma Pro-175/epsilon Thr-176 and gamma Glu-176/epsilon Ala-177 fully account for the 1000-fold selectivity of NmmI for the alpha gamma over the alpha epsilon site.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Assignment of the residues contributing to epsilon  subunit insensitivity to NmmI binding. A, top, the junctions of two chimeras, gamma 173epsilon and gamma 177epsilon . The shaded bar represents gamma  subunit sequence followed the nonshaded epsilon  subunit. Junctional amino acids are shown by the bar. Amino acids from epsilon  are designated in italics. Numbering is for gamma  subunit. Only two differences at positions gamma 175 and 176 (epsilon 176/177) exist in this region. A, bottom, mutations (*) were introduced at gamma  or epsilon  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 gamma  subunit. The superscripts in italics show reported determinants for binding of various ligands. Superscripts are: waglerin, W (30); alpha -conotoxin MI, CM (29); anionic residues for agonists, A (25, 26), and acetylcholine, ACh (36).

Sequence alignment shows that the delta  subunit contains the same residues in this region as the gamma  subunit, consistent with high affinity of the alpha delta site. We therefore attempted to produce a low affinity alpha delta site by introducing the residue determinants in epsilon  that reduce gamma  affinity into equivalent positions of the delta  subunit. The mutations delta P181T and delta E182A, singly or combined, do not affect appreciably NmmI affinity (Table I), indicating that substitutions of other residues unique to the epsilon  subunit into the delta  subunit are required to decrease affinity of NmmI for the alpha delta site.

Residues in NmmI That Interact with Selectivity Determinants in the gamma  and epsilon  Subunits-- Because Pro-175 of the gamma  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 epsilon  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 gamma 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 gamma Glu-176. The receptor mutation gamma E176K decreases affinity of the alpha gamma site for NmmI by nearly 3 orders of magnitude. Similarly, the NmmI mutation K27E decreases affinity of NmmI for the alpha gamma site by approximately 2.5 orders of magnitude. However, combining both gamma 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 gamma E176K and K27E preserves a stabilizing interaction, suggesting that electrostatic force between gamma 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).


View larger version (6K):
[in this window]
[in a new window]
 
Scheme I.  

The loss of energy, Delta Delta G, arising from substitution from wild type into mutant is calculated from the dissociation constant (KD) as follows.
&Dgr;&Dgr;G=RT <UP>ln</UP><FR><NU>K<SUB>D,<UP>mt</UP></SUB></NU><DE>K<SUB>D,<UP>wt</UP></SUB></DE></FR> (Eq. 1)
The coupling energy, Delta Delta GINT, is defined in terms of the respective dissociation constants (K) of the complexes,
   &Dgr;&Dgr;G<SUB><UP>INT</UP></SUB>=RT <UP>ln</UP><FR><NU>K<SUB>R*T*</SUB>×K<SUB>RT</SUB></NU><DE>K<SUB>RT*</SUB>×K<SUB>R*T</SUB></DE></FR>=RT <UP>ln</UP><FR><NU>K<SUB>R*T*</SUB></NU><DE>K<SUB>RT*</SUB></DE></FR>−RT <UP>ln</UP><FR><NU>K<SUB>R*T</SUB></NU><DE>K<SUB>RT</SUB></DE></FR> (Eq. 2)
=(&Dgr;G°<SUB>R*T*</SUB>−&Dgr;G°<SUB>RT*</SUB>)−(&Dgr;G°<SUB>R*T</SUB>−&Dgr;G°<SUB>RT</SUB>) (Eq. 3)
=(&Dgr;G°<SUB>R*T*</SUB>−&Dgr;G°<SUB>R*T</SUB>)−(&Dgr;G°<SUB>RT*</SUB>−&Dgr;G°<SUB>RT</SUB>) (Eq. 4)
where the Delta 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 gamma 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 gamma Glu-176 of the binding site, with the most proximal charged residue being Lys-27 of the toxin.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The alpha -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 alpha -subunit of the receptor retains the capacity to bind alpha -BgTx, whereas isolated beta , gamma , or delta  subunits do not, the alpha -toxins bind with far lower affinity to the alpha  subunit than to the intact receptor. Moreover, small agonists and antagonists do not compete with alpha -toxin binding to the isolated alpha  subunit at expected concentrations (23). These observations point to a predominant, but not sole, contribution to the alpha -neurotoxin binding coming from the alpha  subunit. Our previous work showed that Val-188, Tyr-190, Pro-197, and Asp-200 of alpha  subunit contribute to NmmI binding (19). Also glycosylation at positions 189 and 187, yielding oligosaccharides uniquely found in cobra and mongoose nAChR, reduced alpha -BgTx binding substantially (16).

Although the alpha  subunit appears to be the predominant site of alpha -toxin binding, chemical cross-linking and mutagenesis studies show that non-alpha subunits are close to the site of alpha -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-alpha subunits in contributing to high affinity alpha -toxin binding, as NmmI binds to alpha epsilon interfaces of the adult type of nAChR (alpha 2beta epsilon delta ) with 3 orders of magnitude lower affinity than to the alpha gamma and alpha delta interfaces of the fetal receptor (alpha 2beta gamma delta ). Binding studies, initially using chimeras and subsequently point mutants, show that epsilon Thr-176/epsilon Ala-177 (Pro/Glu in gamma /delta ) contribute entirely to insensitivity of the alpha epsilon interface to NmmI. The observation that alpha -bungarotoxin association is only slightly affected by the gamma  and epsilon  sequence differences suggests that this region of the gamma , epsilon , and delta  subunits is not used equivalently for stabilization of the entire family of bound alpha -neurotoxins. At the present time, it is unclear whether stabilization from this region is unique to some of the short alpha -neurotoxins, or the long alpha -neurotoxins, such as alpha -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 alpha 7 subtype of nAChR (24).

Residues at the gamma 175/176 positions were previously unrecognized as determinants of ligand binding. However, they are immediately adjacent to gamma Asp-174, which was shown by cross-linking to be ~9 Å away from Cys-192/193 in the alpha  subunit (25, 26) and was shown to influence the affinity of quaternary agonists and antagonists (27, 28). Moreover, the adjacent residues of gamma Phe-172 (delta Asp-178, epsilon Ile-173) are known to confer site-selectivity to the smaller competitive peptide inhibitors such as alpha -conotoxin MI (29) and waglerin (30). The equivalent region of the alpha 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 alpha  subunit interface of the binding site, both aromatic (Tyr-190, Tyr-198) and anionic (Asp-200) residues were mapped to the alpha -toxin binding surface (19). Here, we identify another anionic residue in the gamma  subunit, Glu-176, perhaps restricted in its position by a neighboring secondary amino acid Pro-175, on the gamma  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 gamma  subunit (cf. Fig. 5). In this analysis, the loss of free energy (Delta Delta G) associated with a single charge mutation results from all interactions between the charged residue and its multiple neighboring residues. The pairwise interactions (Delta Delta 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, Delta Delta GINT from Equation 2 should largely reflect the Coulombic interaction between the respective paired residues (32).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 5.   Structure of an alpha -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.

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 gamma  and delta  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 gamma  and delta  subunits in preference to the alpha  subunit (13). Thus mutagenesis and chemical labeling studies showed a crucial role of lysine at position 27 and its proximity to gamma  and delta  subunits. Here, our mutant cycle studies delineate the interaction between Glu-176 of the gamma  subunit and Lys-27 of NmmI toxin. The largest linkage in loop II between K27E and gamma E176K (Delta Delta G = -5.9kcal/mol) and smaller linkages (Delta Delta GINT = -1.6 to -3.0 kcal/mol) found previously between K27E and alpha  subunit residues of Val-188, Tyr-190, Pro-197, and Asp-200 (35) correlate well with the labeling studies. A more complete elucidation of alpha -toxin-receptor interactions should enable us to orient a docked alpha -toxin with respect to the subunit interfaces, as well as refine existing models of the structure of the extracellular domain of the receptor (4).


View larger version (17K):
[in this window]
[in a new window]
 
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 alpha -bungarotoxin binding to cell surface nAChR expressed as alpha 2beta gamma delta by wild type NmmI (A) and K27E mutant NmmI (B). C and D, inhibition of the initial rate of 125I-labeled alpha -bungarotoxin binding to cell surface nAChR expressed as alpha 2beta (gamma E176K)delta by wild type (C) and K27E mutant (D) NmmI alpha -toxin. The dashed line in D is the predicted curve when the coupling energy (Delta Delta GINT) between gamma E176K and K27E is 0. The deviation of observed affinity (D) from that predicted by no linkage between the residues at the alpha gamma 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.

Dagger Recipient of a Uehara Memorial Foundation Fellowship.

§ Recipient of a California Tobacco Related Disease Research Program Fellowship.

parallel 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; alpha -BgTx, alpha -bungarotoxin; wt, wild type; mt, mutant.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Unwin, N. (1993) J. Mol. Biol. 229, 1101-1124[CrossRef][Medline] [Order article via Infotrieve]
  2. Karlin, A., and Akabas, M. H. (1995) Neuron 15, 1231-1244[Medline] [Order article via Infotrieve]
  3. Hucho, F., Tsetlin, V. I., and Machold, J. (1996) Eur. J. Biochem. 239, 539-557[Abstract]
  4. Tsigelny, I., Sugiyama, N., Sine, S. M., and Taylor, P. (1997) Biophys. J. 73, 52-66[Abstract]
  5. Chang, C. C., and Lee, C. Y. (1963) Arch. Int. Pharmacodyn. Ther. 144, 241-257[Medline] [Order article via Infotrieve]
  6. Changeux, J. P., Kasai, M., and Lee, C. Y. (1970) Proc. Natl. Acad. Sci. U. S. A. 67, 1241-1247[Abstract]
  7. Endo, T., and Tamiya, N. (1987) Pharmacol. Ther. 34, 403-451[Medline] [Order article via Infotrieve]
  8. Witzemann, V., Muchmore, D., and Raftery, M. A. (1979) Biochemistry 18, 5511-5518[Medline] [Order article via Infotrieve]
  9. Hamilton, S. L., Pratt, D. R., and Eaton, D. C. (1985) Biochemistry 24, 2210-2219[Medline] [Order article via Infotrieve]
  10. 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]
  11. 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]
  12. Machold, J., Weise, C., Utkin, Y., Tsetlin, V., and Hucho, F. (1995) Eur. J. Biochem. 234, 427-430[Abstract]
  13. 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]
  14. Oswald, R. E., and Changeux, J. P. (1982) FEBS Lett. 139, 225-229[CrossRef][Medline] [Order article via Infotrieve]
  15. Arias, H. R. (1997) Brain Res. Rev. 25, 133-191[CrossRef][Medline] [Order article via Infotrieve]
  16. Kreienkamp, H.-J., Sine, S. M., Maeda, R. K., and Taylor, P. (1994) J. Biol. Chem. 269, 8108-8114[Abstract/Free Full Text]
  17. Sine, S. M. (1997) J. Biol. Chem. 272, 23521-23527[Abstract/Free Full Text]
  18. Marchot, P., Frachon, P., and Bougis, P. E. (1988) Eur. J. Biochem. 174, 537-542[Abstract]
  19. Ackermann, E. J., and Taylor, P. (1997) Biochemistry 36, 12836-12844[CrossRef][Medline] [Order article via Infotrieve]
  20. Sine, S. M., and Taylor, P. (1979) J. Biol. Chem. 254, 3315-3325[Abstract]
  21. Sine, S. M., and Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699[Free Full Text]
  22. Horovitz, A., and Fersht, A. R. (1990) J. Mol. Biol. 214, 613-617[Medline] [Order article via Infotrieve]
  23. Gershoni, J. M., Hawrot, E., and Lentz, T. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4973-4977[Abstract]
  24. 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]
  25. Czajkowski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612[Abstract/Free Full Text]
  26. Czajkowski, C., Kaufmann, C., and Karlin, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6285-6289[Abstract]
  27. Martin, M. D., and Karlin, A. (1997) Biochemistry 36, 10742-10750[CrossRef][Medline] [Order article via Infotrieve]
  28. Osaka, H., Sugiyama, N., and Taylor, P. (1998) J. Biol. Chem. 273, 12758-12765[Abstract/Free Full Text]
  29. Sine, S. M., Kreienkamp, H.-J., Bren, N., Maeda, R., and Taylor, P. (1995) Neuron 15, 205-211[Medline] [Order article via Infotrieve]
  30. Molles, B. E., Kline, E. F., Sine, S. M., McArdle, J. J., and Taylor, P. (1998) J. Physiol. (paris) 92, 470[CrossRef] (abstr.)
  31. Galzi, J. L., Bertrand, S., Corringer, P. J., Changeux, J. P., and Bertrand, D. (1996) EMBO J. 15, 5824-5832[Abstract]
  32. Faiman, G. A., and Horovitz, A. (1996) Protein Science 9, 315-316
  33. 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]
  34. 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]
  35. 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]
  36. 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.