Hydrophobic Pairwise Interactions Stabilize α-Conotoxin MI in the Muscle Acetylcholine Receptor Binding Site
2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês
10.1074/jbc.275.17.12692
ISSN1083-351X
Autores Tópico(s)Ion channel regulation and function
ResumoThe present work delineates pairwise interactions underlying the nanomolar affinity of α-conotoxin MI (CTx MI) for the α-δ site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the α2βδ2 pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of125I-α-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for α2βδ2 pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the α subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the δ subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the δ subunit and that δSer-36 and δIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the δ subunit, and between Ala-7 and Pro-6 and Tyr-198 in the α subunit. The overall results reveal the orientation of CTx MI when bound to the α-δ interface and show that primarily hydrophobic interactions stabilize the complex. The present work delineates pairwise interactions underlying the nanomolar affinity of α-conotoxin MI (CTx MI) for the α-δ site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the α2βδ2 pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of125I-α-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for α2βδ2 pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the α subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the δ subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the δ subunit and that δSer-36 and δIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the δ subunit, and between Ala-7 and Pro-6 and Tyr-198 in the α subunit. The overall results reveal the orientation of CTx MI when bound to the α-δ interface and show that primarily hydrophobic interactions stabilize the complex. acetylcholine receptor α-conotoxin MI human embryonic kidney base pair Recent studies have used protein toxins to probe active sites of ligand- and voltage-gated ion channels (1.Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (428) Google Scholar, 2.Naranjo D. Miller C. Neuron. 1996; 16: 123-130Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3.Aiyar J. Withka J. Rizzi J. Singleton D. Andrews G. Lin W. Boyd J. Hanson D. Simon M. Dethlefs B. Lee C. Hall J. Gutman G. Chandy G. Neuron. 1995; 15: 1169-1181Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 4.Chang N. French R. Lipkind G. Fozzard H. Dudley S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (92) Google Scholar, 5.Quiram P.A. Jones J.J. Sine S.M. J. Biol. Chem. 1999; 274: 19517-19525Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 6.Ackerman E.J. Ang E.T.-H. Kanter J.R. Tsigelney I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). By identifying multiple pairwise interactions, these studies define dimensions of the active site according to the known structure of the toxin. The studies also establish the underlying basis for molecular recognition in high affinity protein complexes. Here we probe the muscle AChR1 with the peptide toxin α-conotoxin MI and use double mutant cycles analysis to identify pairs of residues that confer the nanomolar affinity of the complex. Mutagenesis and site-directed labeling studies establish that the ligand binding sites of the muscle AChR are formed at interfaces between α1 and either δ, ε, or γ subunits (7.Prince R.J. Sine S.M. Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Landes Bioscience, Austin, TX1997: 31-59Google Scholar, 8.Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Residues on the α1 face of the binding site are found in three well separated regions of the primary sequence, termed loops A, B, and C. Using the numbering system for the mouse α1subunit, key residues in these loops include Tyr-93 in loop A, Trp-149 in loop B, and Tyr-190 and Tyr-198 in loop C. Similarly, residues on the non-α face of the binding site are found in four well separated regions of the primary sequence, termed loops I through IV. Using the numbering system for the mouse δ subunit, key residues in these loops include Ser-36 in loop I, Trp-57 in loop II, Tyr-113 in loop III, and Ile-178 in loop IV. The observation that these seven loops converge to form a localized binding site has led to a multi-loop model of the major extracellular domain of the AChR (8.Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar). α-Conotoxins are small, disulfide-rich peptides that competitively inhibit muscle and neuronal nicotinic AChRs (9.McIntosh J.M. Santos A.D. Olivera B. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (280) Google Scholar). All α-conotoxins have a conformationally constrained two-loop structure formed by two disulfide bridges. However, the various α-conotoxins differ by the number and type of residues in each loop, allowing specific targeting of receptor subtypes. α-Conotoxins specific for muscle AChRs include MI, GI, and SI, and contain three residues in the first loop and five in the second (Fig. 1). Muscle-specific α-conotoxins can be further subdivided according to their ability to select between the two AChR binding sites; CTx MI and GI select between the two binding sites by 10,000-fold, whereas CTx SI selects between the sites by 100-fold (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 11.Groebe D. Gray W. Abramson S. Biochemistry. 1997; 36: 6469-6474Crossref PubMed Scopus (67) Google Scholar, 12.Hann R.M. Pagan O.R. Eterovic V.A. Biochemistry. 1994; 33: 14058-14063Crossref PubMed Scopus (76) Google Scholar). Moreover, CTx MI binds to the α-δ site of the muscle AChR with nanomolar affinity and stays bound for more than 6 h (13.Prince R.J. Sine S.M. J. Biol. Chem. 1999; 274: 19623-19629Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Their site selectivity and exceedingly high affinity make CTx MI and GI powerful probes of the structure of the muscle AChR binding site. Residues from both α and non-α faces of the AChR binding site stabilize bound CTx MI and include residues from four of the seven loops. The α face contributes Tyr-198 and Tyr-190 from loop C (14.Sugiyama N. Marchot P. Kawanishi C. Osaka H. Molles B. Sine S. Taylor P. Mol. Pharmacol. 1998; 53: 787-794Crossref PubMed Scopus (42) Google Scholar), whereas the δ face contributes Ser-36 from loop I, Tyr-113 from loop III, and Ile-178 from loop IV (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar). Selectivity of CTx MI for the two AChR binding sites owes to residue differences in δ and γ subunits at these three positions and can be transferred from one binding site to the other by exchanging residues at these key positions. That both α and non-α subunits contribute to CTx MI binding suggests that the toxin bridges the subunit interface, whereas the modular exchangeability across γ and δ subunits suggests the key residues contribute directly to CTx MI binding. By mutating residues in both the AChR and CTx MI, the present work further tests the hypothesis that the toxin bridges the binding site interface. We use double mutant cycles analysis to distinguish interacting from non-interacting pairs of residues in the complex. We find that CTx MI interacts with the α-δ site of the AChR through four hydrophobic residues in its N- and C-terminal loops. Furthermore, the key side chains in CTx MI localize in a hydrophobic cluster that interacts with hydrophobic and aromatic residues from both the α and δ subunits. α-Conotoxin MI was purchased from American Peptide Company; 293 human embryonic kidney cell line (293 HEK) and BOSC 23 HEK cell line were from the American Type Culture Collection;125I-labeled α-bungarotoxin was from NEN Life Science Products; d-tubocurarine chloride was from ICN Pharmaceuticals; and 5,5′-dithiobis-2-nitrobenzoic acid was from Sigma. Wild type and mutant α-conotoxin MI were synthesized by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems 431A peptide synthesizer. Cysteine protecting groups (S-triphenylmethyl) were incorporated during synthesis at cysteines 4 and 14, and acetamidomethyl-protecting groups (ACM) were incorporated at cysteines 3 and 8. The linear peptide was purified by a reversed-phase high performance liquid chromatography using a Vydac C18 preparative column with trifluoroacetic acid/acetonitrile buffer. The two disulfide bridges were formed as follows: the cysteineS-triphenylmethyl-protecting groups of cysteines 4 and 14 were removed during trifluoroacetic acid cleavage of the linear peptide from the support resin, and the peptide was oxidized by molecular oxygen to form the 4–14 disulfide bond by stirring in 50 mm ammonium bicarbonate buffer, pH 8.5, at 25 °C for 24 h. The peptide was lyophilized and then the second bridge was formed as follows: the ACM-protecting groups on cysteines 3 and 8 were removed oxidatively by iodine as described (15.Andrue D. Albericio F. Solé N.A. Munson M.C. Ferrer M. Barany G. Methods Mol. Biol. 1994; 35: 139-140Google Scholar), except the peptide/iodine reaction was allowed to progress for 16 h prior to carbon tetrachloride extraction. Residual iodine was separated from the pure product by high performance liquid chromatography. The purified product was verified by mass spectrometry (Table I). The CTx MI mutants are named as follows: the first letter and number refer to the wild type residue and position, and the following letter is the substituted residue at that position.Table IMolecular weights and oxidation states of wild type and mutant α-conotoxinsConotoxinObservedM rCalculated M r% AbsNon-oxidized CTx MI100CTx MI2.5Acetylated G11536.01540.01.4R2Q1465.01465.01.4H5A1428.01430.01.9P6G1454.01453.01.8P6A1467.01467.01.4P6I1509.51510.01.8P6V1495.51497.02.1G9A1494.21498.32.6G9S1494.51497.22.9G9V1472.21474.42.7A7V1466.21467.62.2A7S1467.31468.52.2K10Q1495.01499.02.6K10Y1532.01535.02.1N11K1509.01509.01.7Y12T1432.01432.72.0Y12F1478.01478.03.2S13A1493.01493.02.1S13V1494.01495.02.2Molecular weight was determined by mass spectrometry and compared to the calculated molecular weight. The percent absorbance (% Abs) is the reactivity of each α-conotoxin to DTNB relative to that of linear, non-oxidized α-conotoxin as described under "Experimental Procedures." Commerically available CTx MI is included for comparison. Open table in a new tab Molecular weight was determined by mass spectrometry and compared to the calculated molecular weight. The percent absorbance (% Abs) is the reactivity of each α-conotoxin to DTNB relative to that of linear, non-oxidized α-conotoxin as described under "Experimental Procedures." Commerically available CTx MI is included for comparison. To confirm disulfide bond formation, we compared reactions with Ellman's reagent for linear, non-oxidized CTx MI, commercially available CTx MI, and all of our synthetic CTx MI mutants. For each conotoxin, 100 μg was dissolved in 200 μl of 0.1m phosphate buffer; 4 μl of 5,5′-dithiobis-2-nitrobenzoic acid was added, and the mixture was incubated at room temperature for 30 min. The absorbance at 405 nm was measured. Reactivity for each synthetic mutant is expressed relative to that obtained for the non-oxidized, linear CTx MI (Table I). Mouse AChR subunit cDNAs were subcloned into the cytomegalovirus-based expression vector pRBG4 (16.Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (169) Google Scholar). All mutations of the α subunit were constructed as described previously (17.Sine S.M. Quiram P. Papanikolaou F. Kreienkamp H.-J. Taylor P. J. Biol. Chem. 1994; 269: 8808-8816Abstract Full Text PDF PubMed Google Scholar). The single point mutations δS36K, δY113S, and δI178F, as well as the double mutant δ(S36K/δI178F) and triple mutant δ(S36K/δY113S/δI178F) were constructed as described (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar). The double mutant δ(S36K/δY113S) was constructed by ligation of the 400-bp PflMI-PflMI fragment containing δY113S to the 5100-bpPflMI-PflMI fragment containing the mutation S36K. The double mutant δ(Y113S/δI178F) was constructed by ligation of the 400-bp PflMI-PflMI fragment containing δY113S with the 5100-bp PflMI-PflMI fragment containing δI178F. All constructs were confirmed by dideoxy sequencing. Human embryonic kidney cells (293 HEK) were transfected with mutant or wild type subunit cDNAs using calcium phosphate precipitation as described previously (16.Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (169) Google Scholar). BOSC 23 HEK cells were used in some experiments with low expressing mutant AChRs. AChR subunit cDNAs were combined in the ratio 2:1:2 for α, β, and δ subunits, respectively. After 24 h at 37 °C, the transfected cells were incubated at 31 °C for an additional 48 h. Three days after transfection, intact cells were harvested by gentle agitation in phosphate-buffered saline containing 5 mm EDTA. α-Conotoxin MI binding to intact cells was measured by competition against the initial rate of125I-labeled α-bungarotoxin (18.Sine S.M. Taylor P. J. Biol. Chem. 1979; 254: 3315-3325Abstract Full Text PDF PubMed Google Scholar). After harvesting, the cells were briefly centrifuged, resuspended in potassium Ringer's solution, and divided into aliquots for α-conotoxin binding measurements. Potassium Ringer solution contains 140 mmKCl, 5.4 mm NaCl, 1.8 mm CaCl2, 1.7 mm MgCl2, 25 mm HEPES, and 30 mg/liter bovine serum albumin adjusted to pH 7.4 with 10 mmNaOH. Specified concentrations of α-conotoxin were added 60 min prior to the addition of 125I-α-bungarotoxin, which was allowed to occupy approximately half the surface receptors. Binding was terminated by the addition of 2 ml of potassium Ringer's solution containing 600 μm d-tubocurarine chloride. Cells were harvested through Whatman GF-B filters using a Brandel cell harvester and washed three times with 3 ml of potassium Ringer's solution. Prior to use, the filters were soaked in potassium Ringer's solution containing 4% skim milk for 2 h. Nonspecific binding was determined in the presence of 300 μm d-tubocurarine. The total number of α-bungarotoxin sites was determined by incubation with radiolabeled toxin for 120 min. The initial rate of toxin binding was calculated as described previously to yield the fractional occupancy of the ligand (18.Sine S.M. Taylor P. J. Biol. Chem. 1979; 254: 3315-3325Abstract Full Text PDF PubMed Google Scholar). Binding measurements were analyzed according to the Hill equation: 1 − Y = 1/(1 +([ligand]/K app)n H), where Y is the fractional occupancy of the ligand,K app is the apparent dissociation constant, andn H is the Hill coefficient. For determinations from single experiments, fitted parameters and standard errors were obtained using the program Ultrafit (BIOSOFT). For multiple experiments, means ± S.D. of the individual fitted parameters are presented (Tables II and III).Table IIBinding parameters for CTx MI mutationsK appn HnnmWT CTx MI0.94 ± 0.090.70 ± 0.0230Acetyl-G16.39 ± 0.150.70 ± 0.072R2Q24.21 ± 7.020.80 ± 0.069H5A15.03 ± 5.040.82 ± 0.067P6G7991.02 ± 548.041.30 ± 0.129P6I10550.03 ± 300.261.01 ± 0.052P6A408.15 ± 23.310.91 ± 0.062P6V802.33 ± 14.421.10 ± 0.021A7V6419.24 ± 1239.170.97 ± 0.119A7S46.16 ± 2.440.76 ± 0.043G9A1390.07 ± 239.350.78 ± 0.061G9S4827.32 ± 832.110.82 ± 0.077K10Q27.41 ± 4.190.83 ± 0.097K10Y277.32 ± 28.341.11 ± 0.263N11K28.27 ± 6.100.85 ± 0.087Y12T9902.19 ± 690.341.10 ± 0.0420Y12F2.42 ± 0.340.82 ± 0.081S13A24.16 ± 2.230.92 ± 0.031S13V48.22 ± 3.140.77 ± 0.041Values are the least squares fit to the Hill equation;K app is the apparent dissociation constant;n H is the Hill coefficient, and n is the number of independent experiments. Open table in a new tab Table IIIMutagenesis of the AChR, binding parameters for CTx MIlogKappmutantKappwild typeK appn HnmWTα2βδ20.94 ± 0.090.70 ± 0.02δ subunit mutations S36K1.0610.86 ± 1.090.81 ± 0.03 Y113S1.3119.33 ± 3.430.89 ± 0.05 I178F−0.420.36 ± 0.080.80 ± 0.06 S36K + I178F2.85662 ± 760.81 ± 0.11 S36K + Y113S + I178F4.3119104 ± 37291.01 ± 0.04 S36K + Y13S2.86667 ± 740.70 ± 0.04 Y113S ± I178F−0.200.59 ± 0.030.76 ± 0.03α subunit mutations Y93T−0.530.28 ± 0.060.69 ± 0.02 Y190T1.4828 ± 5.90.80 ± 0.08 Y198T2.77554 ± 781.03 ± 0.06 W149Y0.543.5 ± 0.10.61 ± 0.08 Y151F−0.190.61 ± 0.060.80 ± 0.03K app is the apparent dissociation constant;n H is the Hill coefficient, and n is the number of independent experiments. Results are from experiments shown in Figs. 4 and 5. Open table in a new tab Values are the least squares fit to the Hill equation;K app is the apparent dissociation constant;n H is the Hill coefficient, and n is the number of independent experiments. K app is the apparent dissociation constant;n H is the Hill coefficient, and n is the number of independent experiments. Results are from experiments shown in Figs. 4 and 5. Mutations of CTx MI were generated by standard peptide synthesis methods as described under "Experimental Procedures." Molecular weight and the presence of two disulfide bonds were verified by mass spectrometry and by negative reaction with Ellman's reagent (Table I). Because CTx MI binds 10,000-fold more tightly to the α-δ than to the α-γ interface of the AChR (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar), we measured CTx MI binding to the α2βδ2 pentameric form of the AChR expressed on the surface of 293 HEK cells. As measured by competition against the initial rate of 125I-α-bungarotoxin binding, our synthetic CTx MI bound with a K d of 0.94 nm (Table II) and was indistinguishable from commercially available CTx MI. We mutated all non-cysteines in CTx MI and measured binding of each mutant conotoxin to α2βδ2 pentamers. Our mutagenic scan of CTx MI reveals four residues essential for high affinity binding as follows: Ala-7 and Pro-6 in the N-terminal loop and Tyr-12 and Gly-9 in the C-terminal loop (Fig.2; Table II). Within the N-terminal loop, mutation of Ala-7 to valine decreases affinity nearly 10,000-fold, whereas mutation to serine decreases affinity by 50-fold (Table II), indicating that position 7 requires a side chain that is both small and hydrophobic. Mutation of the adjacent Pro-6 to glycine decreases affinity nearly 10,000-fold, whereas mutation to alanine or valine decreases affinity 400- and 800-fold, respectively, suggesting the need for both restricted rotation of the peptide backbone and hydrophobic contributions at position 6. Within the C-terminal loop of CTx MI, mutation of Tyr-12 to threonine decreases affinity 10,000-fold, whereas mutation to phenylalanine maintains high affinity (Table II), indicating a purely aromatic contribution at position 12. Mutation of Gly-9 to alanine decreases affinity 1400-fold, whereas mutation to serine decreases affinity 5000-fold, indicating a nearly absolute requirement for glycine, which is small and can accommodate unique combinations of φ and ψ bond angles (19.Gouda H. Yamazaki K. Hasagawa J. Kobayashi Y. Nishiuchi Y. Sakakibara S. Hirono S. Biochim. Biophys. Acta. 1997; 1343: 327-334Crossref PubMed Scopus (37) Google Scholar). Surprisingly, the mutants acetyl-G1 and K10Q, which neutralize positive charges previously thought to be essential for CTx MI bioactivity (11.Groebe D. Gray W. Abramson S. Biochemistry. 1997; 36: 6469-6474Crossref PubMed Scopus (67) Google Scholar,12.Hann R.M. Pagan O.R. Eterovic V.A. Biochemistry. 1994; 33: 14058-14063Crossref PubMed Scopus (76) Google Scholar), produce relatively small changes in affinity (Fig. 2). The overall mutagenesis results reveal four energetically equivalent sources of high affinity in CTx MI, making them potential points of interaction with the α-δ binding site. The four bioactive residues of CTx MI map to a restricted region of its three-dimensional structure (Fig. 3; Ref.19.Gouda H. Yamazaki K. Hasagawa J. Kobayashi Y. Nishiuchi Y. Sakakibara S. Hirono S. Biochim. Biophys. Acta. 1997; 1343: 327-334Crossref PubMed Scopus (37) Google Scholar), indicating that the contact surface at the ligand binding site is likely to be small and complementary to the active region of the toxin. Side chains of the bioactive residues occupy corners of an irregular trapezoid 6.4 to 8.0 Å long and 3.9 to 6.6 Å wide, creating a hydrophobic patch in an otherwise hydrophilic peptide. The ring structures of Pro-6 and Tyr-12 stack parallel to each other, whereas Ala-7 protrudes at right angles to Pro-6, and the methylene α-carbon of Gly-9 leaves a pronounced cavity rimmed by hydrophobic side chains. Thus bioactivity of CTx MI owes to a three-fingered hydrophobic structure at one end of the toxin. Aromatic residues in the AChR α subunit are widely recognized to contribute to ligand affinity (7.Prince R.J. Sine S.M. Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Landes Bioscience, Austin, TX1997: 31-59Google Scholar). We therefore examined the key aromatic residues, Tyr-93, Tyr-190, Tyr-198, Trp-149, and Tyr-151, as potential points of interaction with CTx MI. The mutation αY198T decreases affinity 1000-fold, whereas αY190T decreases affinity 30-fold (Fig.4; TableIII). These results, obtained in symmetric α2βδ2 pentamers, are similar to those described for the α-δ site in asymmetric α2βγδ pentamers (14.Sugiyama N. Marchot P. Kawanishi C. Osaka H. Molles B. Sine S. Taylor P. Mol. Pharmacol. 1998; 53: 787-794Crossref PubMed Scopus (42) Google Scholar). Mutation of the remaining aromatic residues, αTyr-93, αTrp-149, and αTyr-151, only weakly affects affinity. Thus αTyr-198 and αTyr-190 are candidates for interaction with CTx MI. Previous work (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar) showed that residue differences at three equivalent positions of the γ and δ subunits confer the 10,000-fold selectivity of CTx MI for the α-δ over the α-γ interface of the AChR. We therefore re-examined these selectivity determinants as potential points of interaction with CTx MI. Single point mutations of the selectivity determinants, δS36K, δY113S, and δI178F, produce only modest changes in affinity for CTx MI (Fig. 5; Table III). However, when S36K and I178F are combined into a single δ subunit, affinity for CTx MI decreases considerably more than with either mutation alone (Fig. 5), indicating that these residues are interdependent in stabilizing CTx MI (10.Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 14.Sugiyama N. Marchot P. Kawanishi C. Osaka H. Molles B. Sine S. Taylor P. Mol. Pharmacol. 1998; 53: 787-794Crossref PubMed Scopus (42) Google Scholar). The remaining combinations of double mutations, (S36K/Y113S) and (Y113S/I178F), produce roughly additive changes in affinity, indicating little interdependence of these selectivity determinants. When mutations of all three selectivity determinants are combined into a single δ subunit, CTx MI affinity falls 10,000-fold to that of low affinity α2βγ2 pentamers (Fig. 5). Thus all three selectivity determinants in the δ subunit are candidates for interaction with CTx MI. Thermodynamic double mutant cycles analysis has been widely used to identify noncovalent interactions between residues within a single protein and between residues joining different proteins (1.Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (428) Google Scholar, 2.Naranjo D. Miller C. Neuron. 1996; 16: 123-130Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3.Aiyar J. Withka J. Rizzi J. Singleton D. Andrews G. Lin W. Boyd J. Hanson D. Simon M. Dethlefs B. Lee C. Hall J. Gutman G. Chandy G. Neuron. 1995; 15: 1169-1181Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 4.Chang N. French R. Lipkind G. Fozzard H. Dudley S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (92) Google Scholar, 5.Quiram P.A. Jones J.J. Sine S.M. J. Biol. Chem. 1999; 274: 19517-19525Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 6.Ackerman E.J. Ang E.T.-H. Kanter J.R. Tsigelney I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,20.Horowitz A. Fersht A. J. Mol. Biol. 1990; 214: 613-617Crossref PubMed Scopus (305) Google Scholar). To generate a mutant cycle for pairs of CTx MI and AChR mutations, dissociation constants are determined for the four possible combinations of wild type (W) and mutant (M) receptors (r) and conotoxins (t): WrWt, MrWt, WrMt, andMrMt. The resulting dissociation constants are then used to calculate a coupling coefficient Ω (20.Horowitz A. Fersht A. J. Mol. Biol. 1990; 214: 613-617Crossref PubMed Scopus (305) Google Scholar) shown in Equation 1,Ω=Kd (WrWt)·Kd (MrMt)Kd (WrMt)·Kd (MrWt)Equation 1 If Ω equals unity the pair of residues does not interact, whereas if Ω deviates from unity the pair of residues interacts. To identify pairs of interacting residues, we focus on residues in the AChR and CTx MI that significantly affect affinity of the complex and then apply double mutant cycles analysis to all possible pairs of receptor-conotoxin mutations. We find the strongest interaction between the triad of selectivity determinants in the δ subunit and Tyr-12 of CTx MI; binding curves for the corresponding mutant cycle are shown in Fig. 6 A. Individually, mutations in either the receptor or the conotoxin decrease affinity by 10,000-fold. However when mutations in both the receptor and conotoxin are examined together, affinity decreases by 100,000-fold, which is 3 orders of magnitude less than predicted if the contributions were additive. Double mutant cycles analysis reveals a coupling coefficient of 1584 for the δ(S36K + Y113S + I178F)/Y12T pair, corresponding to an interaction free energy of 4.3 kcal/mol (TableIV).Table IVOmega values from double mutant cycles analysisδS36K + I178FδS36K + Y113S + I178FδS36KδY113SδI178FαY198TαY190TαY93TR2Q4.012.62.11.62.020.01.01.0H5A1.61.62.52.05.08.01.34.0P6G126.080.01.03.22.0100.01.61.6A7V20.0316.01.61.32.0638.010.01.6G9S10.0126.01.04.02.53.02.01.0K10Q2.03.22.58.05.010.01.66.3N11K1.610.03.22.53.22.03.21.3Y12T126.01584.02.011.83.039.06.310.0 Open table in a new tab Whereas mutant cycles analysis can identify interacting pairs of residues, it can also identify non-interacting pairs of residues, as illustrated for the pair αY198T/N11K (Fig. 6 B). The receptor mutation αY198T decreases affinity by 1000-fold, whereas the CTx MI mutation N11K decreases affinity by 30-fold. When the two mutations are examined together, affinity decreases by 30,000-fold, which is purely additive, demonstrating that αTyr-198 and Asn-11 do not interact. Thus double mutant cycles analysis readily distinguishes interacting from non-interacting pairs of residues in the AChR-CTx MI complex. Applied to the α subunit face of the AChR binding site, mutant cycles analysis reveals that Tyr-198 interacts significantly with the bioactive residues Ala-7, Pro-6, and Tyr-12 in CTx MI (Fig.7 and Table IV). The coupling coefficient for the αY198T/A7V pair is 638 and corresponds to an interaction free energy of 3.8 kcal/mol. The αY198T/P6G pair exhibits a weaker coupling coefficient of 100, perhaps owing to reduction of a joint contact surface formed by Ala-7 and Pro-6 or to increased conformational flexibility of Ala-7 caused by the P6G mutation. The weaker coupling coefficient of 39 for the Y12T/αY198T pair is likely due to an indirect interaction in which the Y12T mutation produces global changes that propagate to either Ala-7 or Pro-6 (Fig. 3), both of which couple strongly to αTyr-198. Alternatively, the Y12T mutation may allow reorientation of the conotoxin due to loss of the interaction between Tyr-12 and the δ subunit (see Fig. 6 Abelow). The fourth bioactive residue in CTx MI, Gly-9, shows a conspicuous lack of coupling to any of the conserved tyrosines in the α subunit. The rank order of coupling to αTyr-198, Ala-7 > Pro-6 > Tyr-12 ≫ Gly-9, suggests that CTx MI binds with Ala-7 opposing Tyr-198 of the α subunit. Applied to the δ subunit face of the AChR binding site, mu
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