Identification of Pairwise Interactions in the alpha -Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles*

Elizabeth J. Ackermann, Eudora T.-H. Ang, Joan R. Kanter, Igor Tsigelny, and Palmer TaylorDagger

From the Department of Pharmacology 0636, University of California, San Diego, La Jolla, California 92093

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha -Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha -neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha -subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha -neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alpha delta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha -subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The nicotinic acetylcholine receptor (nAChR)1 is a member of the large family of neurotransmitter-gated ion channels (for review, see Refs. 1-4). It is composed of five subunits that are arranged in the circular order of alpha gamma alpha delta beta . The two ligand binding sites reside in the extracellular domain at the alpha gamma and alpha delta subunit interfaces (5, 6). A high resolution structure is not available for any member of the family of neurotransmitter-gated ion channels. However, within this family the structure of the nAChR has been most extensively characterized through site-directed labeling (7-12), site-specific mutagenesis (13-23), electron microscopy reconstruction analysis (24, 25), and homology modeling (26). In current models, three discontinuous regions or domains on the alpha -subunit (encompassing residues around 93, 149-154, and 180-200) and four regions on the gamma /delta subunits are thought to participate in the formation of the binding sites (2, 26). Because of sequence differences in the delta  and gamma  subunits, the binding sites on the receptor are not identical; consequently, many ligands bind to each site with different affinities (15, 22, 27).

The critical tool utilized in the initial identification of the receptor and in subsequent structural analyses is the family of three-fingered snake alpha -neurotoxins (for review, see Refs. 28 and 29), which form high affinity complexes with the receptor; for example, the alpha -bungarotoxin-receptor complex has a Kd of ~10-12. The structure of the alpha -neurotoxins has been solved through nuclear magnetic resonance (30-32) and x-ray crystallographic studies (33-35). These polypeptides (~7 kDa) are characterized by three large loops which extend from a rigid globular domain held together by 4 or 5 conserved disulfide bonds. Even though alpha -neurotoxin structures have been solved, little is known about the structure of the toxin-receptor complex, and interacting residues have not been identified.

In previous work, we delineated residues involved in the binding interaction on both the alpha -neurotoxin, Naja mossambica mossambica (NmmI), and the mouse muscle nAChR interfaces (36). Four residues located on the receptor alpha -subunit and three residues located on the toxin structure were found to contribute significantly to high affinity binding. Even though wild-type NmmI displays an equivalent affinity for the two binding sites on nAChR, we showed through mutational analysis that the energetic contribution of selected residues differed at the two sites. Several of the toxin and receptor mutations studied differentially affected binding at the alpha delta and alpha gamma sites, resulting in two distinct binding affinities (36). In this study, we have utilized double mutant cycles to explore whether any of these residues are involved in pairwise interactions at each binding site. Such information may not only lead to a better understanding of the structure of the toxin-receptor complex, but combined with the known structure of the toxin may eventually establish spatial constraints within the receptor architecture (37-39).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 125I-alpha -Bungarotoxin (specific activity ~16 µCi/µg) was obtained from NEN Life Science Products. alpha -Conotoxin M1 was purchased from American Peptide Company.

NmmI Expression and Purification-- Recombinant wild-type and mutant NmmI were expressed as fusion proteins in Escherichia coli, and the free toxins were purified as described (36). Because several of the mutant toxins produced rather low affinity complexes with mutant receptors, relatively large amounts of NmmI alpha -neurotoxins were required for this study. Typically, 0.5-1.5 mg of toxin could be purified to homogeneity from 1 liter of cells.

Receptor Mutagenesis-- All receptor mutants were made as described previously (20, 23, 27, 36, 40).

Expression of Wild-type and Mutant nAChR-- cDNAs encoding the mouse muscle nAChR subunits (alpha , beta , gamma , and delta ) in a cytomegalovirus-based expression vector pRBG4 were co-transfected in a ratio of 2:1:1:1 into HEK 293 cells (at ~50% confluency) using Ca3(PO4)2 precipitation. After 16 h, the medium containing cDNA was replaced with fresh medium (Dulbecco's modified Eagle's medium plus 10% fetal calf serum), and expression was measured 3-4 days after transfection (20).

NmmI Binding Measurements-- Binding assays were carried out on assembled pentameric nAChRs expressed on the surface of intact cells. The cells were harvested by gentle agitation in phosphate-buffered saline plus 5 mM EDTA, centrifuged briefly, and resuspended in high potassium Ringer's solution. The cells were divided into aliquots for binding measurements (assay volume 200 µl). Specified concentrations of NmmI were added to each tube containing receptor and allowed to bind for 5 h. NmmI dissociation constants were measured by competition against initial rates of 125I-alpha -bungarotoxin binding using 10-20 nM concentrations (41).

A concentration of alpha -bungarotoxin considerably above its Kd (60 pM) was used. The assay relies on the fractional reduction of the initial rate of alpha -bungarotoxin binding in the presence and absence of NmmI. alpha -Bungarotoxin rates are less affected by the receptor mutations, and these reductions will not influence the determined NmmI Kd as long as alpha -bungarotoxin dissociation is slow relative to the time frame of the initial rate assay.

Data were analyzed using least squares fits to the Hill equation or to two sites of equal population but different affinities. Nonspecific binding was determined in the presence of 10 mM carbamylcholine, 300 µM dimethyl d-tubocurarine, or 4 µM cobra alpha -toxin (Naja naja siamensus), depending on the particular receptor mutant. Binding assays conducted in the presence of alpha -conotoxin M1 were carried out in an identical manner. Concentrations of alpha -conotoxin M1 utilized for assays with the receptor mutations were as follows: wild type (300 nM), V188D (300 nM), V188K (300 nM), Y190T (1 µM), Y190F (300 nM), P197I (1 µM), and D200Q (1 µM) and were based on previously determined Kd values for alpha -conotoxin M1 (40).

Homology Modeling-- Using erabutoxin b as a template (an alpha -neurotoxin of known crystal structure (33) and possessing 60% residue identity with NmmI), segments of NmmI were modeled on the basis of conserved regions and the common disulfide linkages. The final conformation of the alpha -carbon backbone was adjusted to account for the two unique prolines in erabutoxin b and the single proline in NmmI. The modeled structure was then relaxed by unrestrained steepest descent minimization of 5000 iterations with the program Discover (MSI, 1997).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

nAChR Binding Site-- Of the three segments of linear sequence in the alpha -subunit, the region encompassing residues 180-200 appears to be the most critical for NmmI recognition. Substitutions at residues 188, 190, 197, and 200 resulted in substantial decreases in toxin binding affinity (36), indicating major roles of these residues in alpha -neurotoxin recognition. Determinants in this region include a conserved aromatic residue (Tyr190), two positions where neuronal and muscle receptors differ (Val188 and Pro197), and a negatively charged residue (Asp200). The mutations Y190F and Y190T both resulted in a loss of affinity of ~2 kcal/mol at the alpha delta site and almost 4 kcal/mol at the alpha gamma site (Table I), suggesting the importance of the aromatic hydroxyl group. Introducing a positive charge at position 188 (V188K) was also unfavorable and destabilized the toxin-receptor complex by 1.8 kcal/mol (at the alpha delta site) and 3.5 kcal/mol (at the alpha gamma site), whereas introduction of a negative charge (V188D) at this position had either no effect or far less of an influence (-0.09 kcal/mol at the alpha delta site and 1.8 kcal/mol at the alpha gamma site). The selective loss in binding affinity observed with the introduction of a positive charge but not a negative charge suggested destabilization resulted from coulombic repulsion of the highly cationic alpha -neurotoxin. Elimination of the negative charge at position 200 (D200Q) decreased binding by 66-fold (2.5 kcal/mol) selectively at the alpha gamma site.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Binding and coupling energies

alpha -Neurotoxin Structure and Binding Site-- Sequence comparisons between members of the alpha -neurotoxin family have identified ~12 residues that are highly conserved, three of which are positively charged (Lys27, Arg33, Lys47; NmmI numbering). Extensive mutagenesis studies carried out by Menez and co-workers (42-44) using a homologous alpha -neurotoxin with 60% residue identity to NmmI (erabutoxin a) defined the toxin binding surface to encompass ~680 Å2 and about 10 amino acids located on loop II and the tips of loops I and III. Five of these residues appear to play the predominant role in binding (Ser8, Gln10, Lys27, Arg33, Lys47) (Fig. 1). The binding site utilized for the NmmI toxin appears to be similar yet not identical to that defined for erabutoxin a (36). Whereas Gln10 in erabutoxin a and Glu10 in NmmI affect binding differently, similar to erabutoxin a, the three conserved positive residues are critical for the NmmI-mouse muscle nAChR high affinity interaction.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   Structure of alpha -neurotoxin from N. mossambica mossambica with side chains of mutated residues darkened. Loops I, II, and III of the toxin are labeled. This structure was obtained through an energy minimization with the solved structure of erabutoxin a, which shares a 60% amino acid sequence identity with NmmI. Sequence identities of the two 62-amino-acid peptides are shown.

Homology modeling of NmmI using the solved crystal structure of erabutoxin a as a template is shown in Fig. 1. Locations of each of the residues studied here are indicated. R33E is near the tip of loop II, whereas K27E is closer to the top and opposite side of loop II. K47A is located on an exposed surface of loop III. The mutations K27E and R33E resulted in large changes in binding affinities for the NmmI-nAChR interaction at both the alpha delta and alpha gamma interfaces (Table I). A shift in binding affinity of over 4 orders of magnitude was observed with R33E at the alpha gamma binding interface and nearly 3 orders at the alpha delta binding interface (Table I). The mutation K47A did not exhibit site selectivity for alpha gamma and alpha delta sites.

Double Mutant Cycles-- Double mutant cycle analyses were applied to the three alpha -neurotoxin variants (K27E, R33E, and K47A) and six alpha -subunit nAChR variants (V188D/K, Y190T/F, P197I, and D200Q) described in order to delineate potential pairwise interactions. This method is based on simple additivity or non-additivity of mutations. If two residues are interacting, then the sum of the free energy change of the single mutations will usually not equal the free energy change measured with both mutations (45-47). This is shown by Equation 1:
&Dgr;&Dgr;G<SUB>(X,Y)</SUB>=&Dgr;&Dgr;G<SUB>(X)</SUB>+&Dgr;&Dgr;G<SUB>(Y)</SUB>+&Dgr;&Dgr;G<SUB><UP>INT</UP></SUB> (Eq. 1)
where Delta Delta G(X) represents the change in free energy caused by a mutation at site X on one interacting species relative to its wild type, Delta Delta G(Y) represents the change in free energy caused by a mutation at site Y on the other species relative to its wild type, Delta Delta G(X, Y) represents the change in free energy caused by both mutations when present together, and Delta Delta GINT (coupling energy) is the measure of the interaction of the two components that are mutated. If the two residues are not linked or interacting, Delta Delta GINT will equal 0, and if the two residues are interacting then the value of Delta Delta GINT may be either positive or negative depending on whether the interaction between the mutated residues reduces or enhances affinity (47). Delta Delta GINT can also be described in terms of the equilibrium constants (39):
&Dgr;&Dgr;G<SUB><UP>INT</UP></SUB>=RT<UP>ln</UP> (&OHgr;) (Eq. 2)
where
&OHgr;=<FR><NU>K<SUB>d<SUB>(<UP>WT,WT</UP>)</SUB></SUB> · K<SUB>d<SUB>(Y,X)</SUB></SUB></NU><DE>K<SUB>d<SUB>(WT,<UP>X</UP>)</SUB></SUB> · K<SUB>d<SUB>(<UP>Y</UP>,WT)</SUB></SUB></DE></FR>. (Eq. 3)

Mutant Pairs and Site Selectivity-- Dissociation constants and changes in free energy for each of the mutant pairs analyzed are shown in Table I. Fig. 2 (A and B), shows typical binding curves obtained with the alpha -subunit mutations Y190T and P197I, respectively, when assayed with each of the toxin mutations; the wild-type toxin/wild-type receptor curve is also shown for comparison. As seen in Table I and Fig. 2, many of the mutant pairs result in large reductions in the overall affinity of the toxin-receptor complex. The dissociation constant for the R33E/V188D pair at the alpha gamma binding site was too large to measure precisely but corresponded to a Kd of more than 0.5 mM. The R33E/P197I mutant pair resulted in a loss of affinity of 8.0 kcal/mol (alpha gamma site) or a loss in Kd of 6 orders of magnitude compared with the wild-type/wild-type interaction. Mutant pairs involving the toxin mutation K27E also resulted in large destabilizations ranging from 3.5 to 7.5 kcal/mol.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Equilibrium binding of wild-type and mutant NmmI alpha -neurotoxin to wild-type and mutant nAChR. A, binding of wild-type NmmI to wild-type nAChR (bullet ) and binding of the mutant toxin K47A (black-down-triangle ), K27E (black-triangle), or R33E (black-square) to the nAChR Y190T. B, binding of wild-type NmmI to wild-type nAChR (bullet ) and binding of the mutant toxin K47A (black-down-triangle ), K27E (black-triangle), or R33E (black-square) to nAChR P197I. C, binding of wild-type NmmI alpha -neurotoxin to wild-type nAChR (bullet ) and binding of the toxin-receptor mutant pair K27E/V188D in the absence (black-triangle) or presence (black-square) of 300 nM alpha -conotoxin M1. Binding determinations for NmmI toxins were measured as the fractional reduction in the initial rates of 125I-alpha -bungarotoxin binding in the absence of NmmI (kmax) or in the presence of the indicated amounts of NmmI (kobs). The curves for the wild-type NmmI-wild-type nAChR interaction and for K27E/V188D in the presence of conotoxin are least squares fits to the Hill equation with nH = 1.0. The remaining curves are least squares fits to two binding sites present in equal populations.

As expected by the demonstration of site selectivity conferred by mutations on the alpha -neurotoxin or receptor when analyzed separately, all of the mutant pairs showed two distinct binding affinities presumably arising from the alpha delta and alpha gamma sites. Analysis of the binding curves yielded Hill coefficients ranging from 0.3 to 0.8, indicating the presence of two classes of binding sites. When these curves were fit to a two-site analysis of equal population, differences in affinity between the two sites ranged from 9- to 85-fold.

To confirm that these binding curves reflected disparate affinities at the alpha delta and alpha gamma subunit interfaces and to ascertain which site possessed the high affinity binding, assays were carried out in the presence of alpha -conotoxin M1. alpha -Conotoxin M1 has a more than 10,000-fold selectivity for the alpha delta binding site over the alpha gamma site on the wild-type mouse receptor (Kd,alpha delta = 0.45 nM, Kd,alpha gamma  = 20 µM) (22, 27). alpha -Conotoxin M1 site selectivity is maintained with each of the receptor mutations studied here despite some changes in the absolute Kd values.2 Thus, when alpha -conotoxin M1 is included in the alpha -neurotoxin-receptor binding assays, it protects the alpha delta site nearly completely, with the residual alpha -neurotoxin binding observed only at the alpha gamma site. Fig. 2C shows binding curves of K27E/V188D assayed in the presence and absence of 300 nM alpha -conotoxin M1. In the absence of alpha -conotoxin M1 the curve gives a Hill coefficient of 0.6 with Kd values of 57 nM and 1.8 µM. In the presence of alpha -conotoxin M1, the number of sites decreased by ~50% and the resulting Hill coefficient increased to 0.9, consistent with residual alpha -neurotoxin binding to a single class of sites. Because alpha -conotoxin M1 will preferentially protect the alpha delta site, the observed Kd of 1.3 µM reflects lower affinity binding of K27E at the alpha gamma site. Accordingly, identical experiments were carried out with each of the toxin-receptor pairs presented in Table I. In all cases, higher affinity alpha -neurotoxin binding was found to correspond to the alpha delta site and lower affinity to the alpha gamma site, as indicated in Table I.

Coupling Analysis-- The coupling coefficient Omega  and coupling energy Delta Delta GINT for each toxin-receptor pair (at both the alpha delta and alpha gamma binding sites) were determined using Equations 1 and 2 (Table I and Fig. 3). Two of the mutant pairs studied gave strong coupling energies above 2.0 kcal/mol and seven pairs gave coupling energies of 1.5 kcal/mol or above. Despite very large shifts in the overall affinities of the mutant pairs relative to that of the wild-type toxin-wild-type receptor complex, the majority of the pairs showed simple additivity of free energy within experimental error (coupling energies approaching 0, 0.1-0.5 kcal/mol). Most strikingly, all of the mutant pairs involving K47A gave values very close to Delta Delta GINT = 0, indicating that the introduced receptor mutations and the toxin mutation K47A do not grossly alter the respective protein structures. A few mutant pairs gave values between 1.0 and 1.3 kcal/mol; intermediate energy values in this range are difficult to interpret because of cumulative errors of addition of free energies.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 3.   Plot of the Omega  values calculated according to Equation 2 for each of the mutant pairs at the alpha delta binding site (upper panel) or alpha gamma binding site (lower panel). Note that the scale for Omega  values differs between the two plots. The value for the R33E/V188D pair at the alpha gamma site is >12, with the exact number not determined due to the very large loss in the overall binding affinity.

As can be seen in Table I, the strengths of the observed linkages are not identical at the alpha delta and alpha gamma binding sites (Fig. 3, A and B). Because the energy contributions of the individual residues alone were found to differ at the two sites, a difference in the coupling energy at the two sites might also be expected.

Interactions of Receptor Residue Val188 with Toxin Residues Arg33 and Lys27-- The two strongest interactions were found between the mutant pairs R33E/V188D and K27E/V188D at the alpha delta interface (coupling energies of 2.6 and 2.1 kcal/mol, respectively). The R33E/V188D pair also appeared to have a strong coupling at the alpha gamma interface (>1.5 kcal/mol), but a precise number could not be obtained. The very large overall loss in the binding affinity required an unachievable production level of mutant toxin. On the other hand, no significant coupling was observed at the alpha gamma interface with the K27E/V188D mutant pair containing the other charge substitution studied in loop 2.

Analysis of the direction of the free energy changes observed in the double mutant cycles involving V188D or V188K and the toxin mutations R33E and K27E are consistent with the involvement of coulombic attractions and repulsions between the introduced charged mutations. For example, the single toxin mutation R33E and single receptor mutation V188D resulted in changes in free energy from the wild-type counterpart of 5.7 and 1.8 kcal/mol, respectively (at the alpha gamma interface). The summations of the individual changes in free energy is then 7.5 kcal/mol, which corresponds to a Kd of ~42 µM. However, the observed Kd of >500 µM with the R33E/V188D mutant pair is larger then predicted for non-interacting sites and corresponded to a coupling energy Delta Delta GINT > 1.5 kcal/mol. A larger than predicted Kd might be expected if these two residues are in close apposition in the complex and experience coulombic repulsion from the two introduced proximal negative charges. Conversely, for the R33E/V188K interaction (at the alpha gamma interface) the Kd found in the binding experiment was lower than predicted for additivity, (predicted Kd = 860,000 nM, Delta Delta G = 9.3 kcal/mol, experimental Kd = 63,000 nM, Delta Delta G = 7.7 kcal/mol) corresponding to a Delta Delta GINT = -1.6 kcal/mol. Again, this finding is consistent with coulombic attraction of the introduced negative charge on the toxin (R33E) and positive charge on the receptor (V188K).

The linkages between these mutant pairs were further explored by examining a network of mutant cycles proceeding among positive, neutral, and negative substitutions shown in Fig. 4. Here additional cycles involving R33E with K188D and K27E with K188D are also analyzed. The mutant cycle for R33E/K188D results in a very strong coupling coefficient of more than 100 at both the alpha delta and alpha gamma binding interfaces (2.8 and >3.0 kcal/mol, respectively). This result reveals a previously unobserved strong linkage at the alpha gamma interface. The same analysis carried out with the K27E/K188D mutant pairs also gives roughly equal linkages at the two sites but 10-fold smaller than those observed with R33E with the same receptor mutations.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Network of mutant cycles between toxin position 33 (A) or toxin position 27 (B) with receptor position 188. The values of Kd in nM for each mutant pair are indicated in parentheses with the upper number corresponding to the Kd at the alpha delta site and the lower number for the Kd at the alpha delta site.

Other Interactions-- The toxin-receptor mutant pairs K27E/Y190F, K27E/Y190T, K27E/P197I, and K27E/D200Q all showed coupling energies of 1.5 kcal/mol or higher at the alpha delta binding site with no significant couplings observed at the alpha gamma binding site. Approximately equal coupling values were found with either Y190T or Y190F and the K27E substitution (Table I). Apart from its coupling to position 188 on the receptor, the R33E substitution showed coupling to the Y190T mutation. This was only observed at the alpha gamma binding site.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Using site-directed mutagenesis, we previously identified residues involved in the high affinity interaction between the alpha -neurotoxin NmmI and the mouse muscle nAChR. The goal of this study was to identify specific pairs of interacting residues between the toxin and the receptor utilizing double mutant cycles (39, 47). This method has been successfully developed to identify pairwise interactions between the scorpion toxins and the potassium channel (37-39). In those studies, the known structure of the toxin was utilized to establish spatial locations of residues on the potassium channel of unknown structure.

In theory, if two residues are coupled either directly or through another residue then the effect of the double mutation will not be equal to the sum of the effects of the two single mutations (47, 48). Therefore, if Delta Delta GINT deviates from 0 (or Omega  deviates from unity) the two mutations under study exhibit an interaction. In practice, small Delta Delta GINT values may exist between residues separated by great distances (49). Conversely, coupling energies greater than 1.5 kcal/mol are generally associated with short distances between the two residues under study (46). In our case, the errors associated with binding measurements of two nonequivalent sites located on one receptor molecule ranged from 10 to 20%. Because of the cumulative errors when summing single mutations, the errors associated with our linkage values ranged from 30 to 50%. Therefore, we have only considered coupling energies above 1.5 kcal/mol for our analysis.

Besides direct interactions or interactions mediated through a proximal residue, large deviations from additivity can also occur when gross structural changes in the individual molecules result from the introduced mutations. If a mutation results in a global conformational change then it would be expected to be linked to a large number of residues. Therefore, the lack of linkages with nearby residues becomes a good indication that structural integrity of the interacting molecules has been maintained. In addition, failure to detect an interaction does not exclude the close proximity of the two residues. This may be due to either weak interactions between the two residues or to interactions that are compensatory yielding a minimal net change. This is especially true in the case of mutant cycles in which the reference side chain, typically the naturally occurring amino acid, exhibits a dominant influence (50).

For example, when the naturally occurring side chain Val188 on the common alpha -subunit of two binding sites is converted to both cationic and anionic side chains therein creating two parallel cycles, different coupling energies are achieved at the alpha delta and alpha gamma interfaces (Fig. 4). However, if we coalesce the two cycles by considering a direct substitution from a cationic to an anionic side chain, then differences between the two sites virtually vanish. This suggests the naturally occurring valine common to both small cycles imparts the asymmetry, and the influence of an inserted charge on alpha -neurotoxin binding is similar at both sites. Interactions intrinsic to the reference residue or steric constraints could influence the alpha delta and alpha gamma sites in a differential manner (50). A more appropriate frame of reference might come from a neutral side chain isosteric with substituted residues or a side chain with minimal steric perturbation (alanine).

Of the 36 residue pairs studied here (18 at each of the alpha delta and alpha gamma binding sites), 25 gave Delta Delta G values below 1.0 kcal/mol, indicating simple additivity. These results suggest that the gross structural changes do not occur in the interacting molecules with the introduced mutations. Rather, the relatively few but large coupling energies that were observed support the specific interactions between these residues.

The strongest linkages observed are with the R33/V188 and K27/V188 toxin-receptor pairs. The strength of the coupling observed between these pairs varied at the alpha delta and alpha gamma binding sites and also with the different amino acid substitutions examined. The toxin residue 33 and receptor residue 188 appear to be interacting at both the alpha delta and alpha gamma binding sites with coupling energies as high as 2.8 and >3.0 kcal/mol observed within the network of cycles. Toxin residue 27 and receptor residue 188 also appear to be interacting at both sites but to a lesser extent, as the coupling energies observed between this pair were generally lower than with the 33/188 pair. In contrast, the toxin residue Lys47 did not show any interaction with the receptor residue Val188.

Coupling energies ranging from 1.5 to 1.9 kcal/mol were also found with the toxin residue Lys27 and the three receptor residues (Tyr190, Pro197, and Asp200), all at the alpha delta interface. These results suggest that Tyr190, Pro197, and Asp200 are close enough in the receptor structure each to be interacting with Lys27. Another possibility is that some or all of these observed linkages are mediated through a third residue. The lack of coupling observed between these paired residues at the alpha gamma binding site does not preclude their interaction or their close proximity. However, it does demonstrate that the energetic contributions of Tyr190, Pro197, and Asp200 to toxin binding differ at the two sites. These data also suggest that the toxin is binding with different orientations to the two ligand sites, where interactions with Tyr190, Pro197, and Asp200 are less critical for the alpha gamma site, but further experiments will be necessary to address this point.

An initial model of binding is proposed from these data. The two conserved toxin cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are complexing with key receptor residues located on the alpha -subunit region between 180 and 200. More specifically, we suggest that the toxin residue Arg33 is adjacent to the receptor residue Val188 and is probably stabilized by adjoining negative or aromatic residues located on the receptor structure. One such candidate may be Tyr190, which did show a linkage with Arg33 at the alpha gamma binding site. Other possibilities include residues located on the delta /gamma subunits (see below). Lys27 also appears to be positioned in the vicinity of Val188 but closer to the residues Tyr190, Pro197, and Asp200. In this case, the lysine cation on the toxin may be directly stabilized through electrostatic interactions with Asp200 and cation/pi interactions with Tyr190.

The involvement of cationic residues near the tip of loop 2 on the toxin and the receptor sequence between residues 180 and 200 has been implicated from single residue mutations, chemical labeling, and binding of toxin to receptor peptide fragments (50-55), but previous studies have not pinpointed specific residue interactions nor have they distinguished differences in alpha -neurotoxin binding between the two binding sites. Homology modeling (26) and labeling experiments (12) have indicated that residues 180-200 on the alpha -subunit are located at the interface formed at the alpha delta and alpha gamma ligand binding sites. Therefore, besides the alpha -subunit residues studied here, it is likely that the toxin loop II residues are interacting with delta /gamma subunit residues. On the other hand, K47A, which is located on loop III of the toxin structure, does not appear to be interacting with this area of the alpha -subunit. Further studies aimed at identifying linkages between toxin residues and receptor residues on the delta  and gamma  subunits should provide the additional constraints necessary to describe the toxin orientation and positioning at the two receptor binding sites.

    ACKNOWLEDGEMENTS

We thank Francisco Lio for technical assistance with the binding assays and Dr. David A. Johnson (University of California, Riverside) for providing cobra alpha -toxin.

    FOOTNOTES

* This work was supported by United States Public Health Service Fellowship NS 10082 (to E. J .A.) and United States Public Health Service Grant GM 18360 (to P. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology 0636, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 619-534-1366; Fax: 619-534-8248; E-mail: priley{at}ucsd.edu.

1 The abbreviations used are: nAChR, nicotinic acetylcholine receptor; NmmI, alpha -neurotoxin I from Naja mossambica mossambica.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Unwin, N. (1993) Cell 72, 31-41[Medline] [Order article via Infotrieve]
  2. Galzi, J.-L., and Changeux, J.-P. (1994) Curr. Opin. Struct. Biol. 4, 554-565
  3. Karlin, A., and Akabas, M. H. (1995) Neuron 15, 1231-1244[Medline] [Order article via Infotrieve]
  4. Hucho, F., Tsetlin, V. I., and Machold, J. (1996) Eur. J. Biochem. 239, 539-557[Abstract]
  5. Blount, P., and Merlie, J. P. (1989) Neuron 3, 349-357[CrossRef][Medline] [Order article via Infotrieve]
  6. Sine, S. M., and Claudio, T. (1991) J. Biol. Chem. 266, 19369-19377[Abstract/Free Full Text]
  7. Kao, P. N., Dwork, A. J., Kaldany, R. J., Silver, M. L., Wideman, J., Stein, S., and Karlin, A. (1984) J. Biol. Chem. 259, 11662-11665[Abstract/Free Full Text]
  8. Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J.-Y., Lazure, C., Chretien, M., and Changeux, J.-P. (1988) Biochemistry 27, 2346-2357[Medline] [Order article via Infotrieve]
  9. Abramson, S. N., Li, Y., Culver, P., and Taylor, P. (1989) J. Biol. Chem. 264, 12666-12672[Abstract/Free Full Text]
  10. Galzi, J.-L., Revah, F., Black, D., Goeldner, M., Hirth, C., and Changeux, J.-P. (1990) J. Biol. Chem. 265, 10430-10437[Abstract/Free Full Text]
  11. Middleton, R. E., and Cohen, J. B. (1991) Biochemistry 30, 6987-6997[Medline] [Order article via Infotrieve]
  12. Czajkowski, C., and Karlin, A. (1995) J. Biol. Chem. 270, 3160-3164[Abstract/Free Full Text]
  13. Tomaselli, G. F., McLaughlin, J. T., Jurman, M. E., Hawrot, E., and Yellen, G. (1991) Biophys. J. 60, 721-727[Abstract]
  14. O'Leary, M. E., and White, M. M. (1992) J. Biol. Chem. 267, 8360-8365[Abstract/Free Full Text]
  15. Sine, S. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9436-9440[Abstract]
  16. Aylwin, M. L., and White, M. M. (1994) Mol. Pharmacol. 46, 1149-1155[Abstract]
  17. Aylwin, M. L., and White, M. M. (1994) FEBS Lett. 349, 99-103[CrossRef][Medline] [Order article via Infotrieve]
  18. Fu, D.-X., and Sine, S. M. (1994) J. Biol. Chem. 269, 26152-26157[Abstract/Free Full Text]
  19. O'Leary, M. E., Filatov, G. N., and White, M. M. (1994) Am. J. Physiol. 266, C648-C653[Abstract/Free Full Text]
  20. Sine, S. M., Quiram, P., Papanikolaou, F., Kreienkamp, H.-J., and Taylor, P. (1994) J. Biol. Chem. 269, 8808-8816[Abstract/Free Full Text]
  21. Nowak, M. W., Kearney, P. C., Sampson, J. R., Saks, M. E., Labarca, C. G., Silverman, S. K., Zhong, W., Thorson, J., Abelson, J. N., Davidson, N., Schultz, P. G., Dougherty, D. A., and Lester, H. A. (1995) Science 268, 439-442[Medline] [Order article via Infotrieve]
  22. Sine, S. M., Kreienkamp, H.-J., Bren, N., Maeda, R., and Taylor, P. (1995) Neuron 15, 205-211[Medline] [Order article via Infotrieve]
  23. Sugiyama, N., Boyd, A. E., and Taylor, P. (1996) J. Biol. Chem. 271, 26575-26581[Abstract/Free Full Text]
  24. Unwin, N. (1993) J. Mol. Biol. 229, 1101-1124[CrossRef][Medline] [Order article via Infotrieve]
  25. Unwin, N. (1996) J. Mol. Biol. 257, 586-596[CrossRef][Medline] [Order article via Infotrieve]
  26. Tsigelny, I., Sugiyama, N., Sine, S. M., and Taylor, P. (1997) Biophys. J. 73, 52-66[Abstract]
  27. Kreienkamp, H.-J., Sine, S. M., Maeda, R. K., and Taylor, P. (1994) J. Biol. Chem. 269, 8108-8114[Abstract/Free Full Text]
  28. Endo, T., and Tamiya, N. (1987) Pharmacol. Ther. 34, 403-451[Medline] [Order article via Infotrieve]
  29. Menez, A. (1991) in Snake Toxins (Harvey, A. L., ed), pp. 35-90, Pergamon Press, New York
  30. Basus, V. J., Billetet, M., Love, R. A., Stroud, R. M., and Kuntz, I. D. (1988) Biochemistry 27, 2763-2771[Medline] [Order article via Infotrieve]
  31. Hatanaka, H., Oka, M., Kohda, D., Tate, S.-I., Suda, A., Tamiya, N., and Inagaki, F. (1994) J. Mol. Biol. 240, 155-166[CrossRef][Medline] [Order article via Infotrieve]
  32. Peng, S.-S., Kumar, T. K. S., Jayaraman, G., Chang, C.-C., and Yu, C. (1997) J. Biol. Chem. 272, 7817-7823[Abstract/Free Full Text]
  33. Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko, A. D., and Richardson, J. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2991-2994[Abstract]
  34. Agard, D. A., and Stroud, R. M. (1982) Acta Crystallogr. A 38, 186-194[CrossRef]
  35. Arnoux, B., Menez, R., Drevet, P., Boulain, J.-C., Ducruix, A., and Menez, A. (1994) FEBS Lett. 342, 12-14[CrossRef][Medline] [Order article via Infotrieve]
  36. Ackermann, E. J., and Taylor, P (1997) Biochemistry 36, 12836-12844[CrossRef][Medline] [Order article via Infotrieve]
  37. Ranganathan, R., Lewis, J. H., and MacKinnon, R. (1996) Neuron 16, 131-139[Medline] [Order article via Infotrieve]
  38. Naranjo, D., and Miller, C. (1996) Neuron 16, 123-130[Medline] [Order article via Infotrieve]
  39. Hidalgo, P., and MacKinnon, R. (1995) Science 268, 307-310[Medline] [Order article via Infotrieve]
  40. Sugiyama, N., Marchot, P., Kawanishi, C., Osaka, H., Molles, B., Sine, S. M., and Taylor, P. (1998) Mol. Pharmacol., in press
  41. Sine, S., and Taylor, P. (1979) J. Biol. Chem. 254, 3315-3325[Abstract]
  42. Pillet, L., Tremeau, O., Ducancel, F., Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Menez, A. (1993) J. Biol. Chem. 268, 909-916[Abstract/Free Full Text]
  43. Tremeau, O., Lemaire, C., Drevet, P., Pinkasfeld, S., Ducancel, F., Boulain, J.-C., and Menez, A. (1995) J. Biol. Chem. 270, 9362-9369[Abstract/Free Full Text]
  44. Ducancel, F., Merienne, K., Fromen-Romano, C., Tremeau, O., Pillet, L., Drevet, P., Zinn-Justin, S., Boulain, J.-C., and Menez, A. (1996) J. Biol. Chem. 271, 31345-31353[Abstract/Free Full Text]
  45. Carter, P. J., Winter, G., Wilkinson, A. J., and Fersht, A. R. (1984) Cell 38, 835-840[Medline] [Order article via Infotrieve]
  46. Schreiber, G., and Fersht, A. R. (1995) J. Mol. Biol. 248, 478-486[CrossRef][Medline] [Order article via Infotrieve]
  47. Wells, J. (1990) Biochemistry 29, 8509-8517[Medline] [Order article via Infotrieve]
  48. Horovitz, A., and Fersht, A. R. (1990) J. Mol. Biol. 214, 613-617[Medline] [Order article via Infotrieve]
  49. LiCata, V. J., and Akers, G. K. (1995) Biochemistry 34, 3133-3139[Medline] [Order article via Infotrieve]
  50. Faiman, G. A., and Horovitz, A. (1996) Protein Eng. 9, 315-316[Abstract]
  51. McLane, K. E., Wu, X., Diethelm, B., and Conti-Tronconi, B. M. (1991) Biochemistry 30, 4925-4934[Medline] [Order article via Infotrieve]
  52. Basus, V. J., Song, G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298[Medline] [Order article via Infotrieve]
  53. Chaturvedi, V., Donnelly-Roberts, D. L., and Lentz, T. L. (1993) Biochemistry 32, 9570-9576[Medline] [Order article via Infotrieve]
  54. Fulachier, M.-H., Mourier, G., Cotton, J., Servent, D., and Menez, A. (1994) FEBS Lett. 338, 331-338[CrossRef][Medline] [Order article via Infotrieve]
  55. Barchan, D., Ovadia, M., Kochva, E., and Fuchs, S. (1995) Biochemistry 34, 9172-9176[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.