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• W2024489413 abstract "Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist,d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR γ-subunit. Because data for mutations at residue γTyr117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of γTyr117 with both the 2-N and the 13′-positions of dTC. Examination of a dTC analog with a negative charge at the 13′-position failed to reveal electrostatic interaction with charged side-chain substitutions at γ117, but the effects of side-chain substitutions remained consistent with proximity of Tyr117 to the cationic 2-N of dTC. The apparent interaction of γTyr117with the 13′-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-π electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to γTyr117. This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR. Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist,d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR γ-subunit. Because data for mutations at residue γTyr117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of γTyr117 with both the 2-N and the 13′-positions of dTC. Examination of a dTC analog with a negative charge at the 13′-position failed to reveal electrostatic interaction with charged side-chain substitutions at γ117, but the effects of side-chain substitutions remained consistent with proximity of Tyr117 to the cationic 2-N of dTC. The apparent interaction of γTyr117with the 13′-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-π electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to γTyr117. This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR. nicotinic acetylcholine receptor acetylcholine-binding protein fromL. stagnalis α-bungarotoxin α-conotoxin MI d-tubocurarine high performance liquid chromatography polyethyleneimine phosphate-buffered saline wild type The muscle nicotinic acetylcholine receptor (AChR)1 is a member of the ligand-gated ion channel superfamily. It is a pseudo-symmetric pentamer with a subunit stoichiometry of α2βγδ. The agonist-binding sites, which are responsible for activating channel opening, lie at the interfaces between the α- and γ-subunits and between the α- and δ-subunits (1Pedersen S.E. Cohen J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2785-2789Crossref PubMed Scopus (225) Google Scholar). The two sites are similar and share conserved features contributed by their respective α-subunits as well as the amino acids that remain constant among the homologous γ- and δ-subunits. The γ- and δ-subunits, however, also determine affinity differences of the two sites for various ligands. Affinity differences between the sites extend to both agonists, such as epibatidine (2Prince R.J. Sine S.M. J. Biol. Chem. 1998; 273: 7843-7849Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and carbamylcholine (3Blount P. Merlie J.P. Neuron. 1989; 3: 349-357Abstract Full Text PDF PubMed Scopus (228) Google Scholar), and to competitive antagonists, such as d-tubocurarine (4Neubig R.R. Cohen J.B. Biochemistry. 1979; 18: 5464-5475Crossref PubMed Scopus (181) Google Scholar). Identification of binding site residues has been carried out by affinity and photoaffinity labeling, by cross-linking studies, by analysis of expressed chimeric receptors, and by site-directed mutagenesis (5Corringer P.J., Le Novere N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (708) Google Scholar). These approaches have found that the binding sites consist of residues from two subunits from several regions (also referred to as loops) that are distant in the linear subunit sequences. Loop c in the α-subunit includes residues α184–α198, which constitute a significant part of the binding determinant for α-bungarotoxin (6Wilson P.T. Hawrot E. Lentz T.L. Mol. Pharmacol. 1988; 34: 643-650PubMed Google Scholar). It also includes an unusual, highly conserved vicinal disulfide bond between αCys192 and αCys193 (7Kao P.N. Karlin A. J. Biol. Chem. 1986; 261: 8085-8088Abstract Full Text PDF PubMed Google Scholar) and several conserved tyrosine residues (αTyr190 and αTyr198). The two other regions within the α-subunit include the residues αTyr93 (8O'Leary M.E. Filatov G.N. White M.M. Am. J. Physiol. 1994; 266: C648-C653Crossref PubMed Google Scholar, 9Cohen J.B. Sharp S.D. Liu W.S. J. Biol. Chem. 1991; 266: 23354-23364Abstract Full Text PDF PubMed Google Scholar) and αTrp149 (10Dennis M. Giraudat J. Kotzyba-Hibert F. Goeldner M. Hirth C. Chang J.Y. Lazure C. Chretien M. Changeux J.P. Biochemistry. 1988; 27: 2346-2357Crossref PubMed Scopus (249) Google Scholar), respectively. The latter residue appears particularly important for interaction with agonist ammonium moieties (11Zhong W. Gallivan J.P. Zhang Y., Li, L. Lester H.A. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12088-12093Crossref PubMed Scopus (499) Google Scholar). Studies addressing the binding site contributions from the γ- and δ-subunits have likewise found contributions from sequence-separated regions of amino acids. Amino acid γTrp55 (12Chiara D.C. Middleton R.E. Cohen J.B. FEBS Lett. 1998; 423: 223-226Crossref PubMed Scopus (46) Google Scholar) lies within one region. A second region includes residues γLeu109–γTyr117. The alternating pattern of residues contributing to binding in this region led to the proposal that the sequence makes a hairpin turn in their vicinity of the binding site (13Chiara D.C. Xie Y. Cohen J.B. Biochemistry. 1999; 38: 6689-6698Crossref PubMed Scopus (59) Google Scholar). Several other γ-subunit residues dispersed in the sequence contribute to ligand binding, either directly or through allosteric effects. They include γLys34, γSer161, γPhe172, and γAsp174(14Martin M. Czajkowski C. Karlin A. J. Biol. Chem. 1996; 271: 13497-13503Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Sine S.M. Kreienkamp H.J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 16Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). The detailed interactions between ligands and this sizable set of amino acid residues are largely unknown. However, the prevalence of aromatic amino acids as well as detailed studies using unnatural amino acid substitution support the hypothesis that cation π-electron interaction are critical for stabilizing the ammoniums of agonists (11Zhong W. Gallivan J.P. Zhang Y., Li, L. Lester H.A. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12088-12093Crossref PubMed Scopus (499) Google Scholar,17Nowak 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. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (213) Google Scholar). Such studies also yielded observations on the possible interactions of several other aromatic residues; Nowak et al. (17Nowak 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. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (213) Google Scholar) showed that αTyr93 is likely to act as a hydrogen bond donor, whereas the aromatic ring of αTyr198appears to interact with the quaternary ammonium of acetylcholine. Antagonists such as dTC have been utilized to study binding site interactions, partly because of the significant affinity difference between the two binding sites. dTC has ∼100–500-fold higher affinity for the αγ site than the αδ site (4Neubig R.R. Cohen J.B. Biochemistry. 1979; 18: 5464-5475Crossref PubMed Scopus (181) Google Scholar). Nonetheless, α-subunit residues also affect binding of dTC. αTyr198 has a strong impact on dTC affinity; when mutated to αY198F, the AChR displayed ∼10-fold greater affinity for dTC, but the mutation had little effect on acetylcholine affinity or efficacy (18Filatov G.N. Aylwin M.L. White M.M. Mol. Pharmacol. 1993; 44: 237-241PubMed Google Scholar). This suggests a unique interaction between the αTyr198 hydroxyl and dTC. Because mutations at αTyr198 and γTyr117 had similar effects on metocurine (sometimes referred to as dimethyl-tubocurarine) affinity, it was proposed that these amino acids each stabilize one of the two quaternary ammoniums on metocurine through cation-π electron interactions (19Fu D.X. Sine S.M. J. Biol. Chem. 1994; 269: 26152-26157Abstract Full Text PDF PubMed Google Scholar). The recent atomic resolution structure of the acetylcholine-binding protein from Lymnaea stagnalis (AChBP), a protein homologous to the N-terminal, ligand-binding domain of the AChR, shows that many of the residues implicated by the studies listed above are in proximity to a binding pocket where they can contribute to stabilization of binding of agonists and antagonists (20Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1586) Google Scholar). The structure included a solvent Hepes molecule that indicates the likely binding locus. The structure is in accord with many of the prior observations regarding ligand-binding site structure and substantially refined the current thinking about ligand-receptor interactions. However, the details of the interactions with cholinergic ligands remain to be fully elucidated, as do the conformational changes that correlate with channel opening and desensitization. Our previous studies (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 22Papineni R.V. Pedersen S.E. J. Biol. Chem. 1997; 272: 24891-24898Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) have taken advantage of dTC analogs to examine the importance of various functional groups to binding interactions and conformational transitions. The structures of the analogs are shown in Fig. 1. For both mouse and TorpedoAChR, we demonstrated that the stereochemistry at the 1 carbon was important for high affinity binding but that the ammoniums at the 2- and 2′-positions need not be quaternary for high affinity binding (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Furthermore, dTC ring D, which includes the 12′- and 13′-positions, interacts in a site-selective manner, consistent with a possible interaction with the γ- and δ-subunits. 13′-Modification further altered the propensity of dTC to desensitize the TorpedoAChR, an observation that may serve as a clue for the structural changes that occur upon conformational shifts of the AChR. In order to pinpoint such interactions between a ligand functional group and a particular amino acid residue, ligand binding energies must be examined in concert with receptor mutagenesis. In this study, we utilize a double mutant thermodynamic cycle analysis to analyze the interaction between our library of dTC analogs (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and residues in the AChR-binding site, with particular emphasis on the interaction with residue γTyr117. We demonstrate that both the 2-N and the 13′-positions of dTC appear to interact with this amino acid but with a proximal interaction only to the 2-N. Based on this conclusion and using the structure of the AChBP, we present a plausible structural model for the orientation and position of dTC within the binding pocket. The α-subunit cDNA clone of the mouse nicotinic AChR was a gift from Dr. Mike White (MCP Hahnemann University); the β-, γ-, and δ-subunit cDNAs were gifts from Dr. James Patrick (Baylor College of Medicine). Restriction enzymes were purchased from Invitrogen or from New England Biolabs (Beverly, MA). The QuickChange mutagenesis kit was obtained from Stratagene (La Jolla, CA), and Endo-free Mega and Giga kits were obtained from Qiagen (Valencia, CA). HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). PEI was obtained either from Miles or from Aldrich. 125I-α-Bungarotoxin was obtained from Amersham Biosciences. Whatman GF/C filters were obtained through VWR Scientific (Houston, TX). All other chemicals were obtained from Sigma or from standard sources. Synthesis of most of the d-tubocurarine analogs was described previously (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Synthesis of several new analogs is described as follows. For 13′-sulfo-d-tubocurarine (sulfo-dTC, 7′,12′-dihydroxy-6,6′-dimethoxy-13′-sulfonate-2,2′,2′-trimethyltubocuraranium trifluoroacetate), 1 g of dTC was treated with 5 ml of concentrated H2SO4 on ice for 3 h. The product was precipitated by dilution with 500 ml of cold ether and collected by centrifugation. The product was purified by cation exchange chromatography over a CM-25 Sephadex column (AmershamBiosciences) and eluted with a gradient of 50–400 mmammonium acetate, pH 6.8. Pure fractions were collected and lyophilized with a net yield of 58%. The material was desalted on a reversed phase Beckman C-18 Ultrasphere preparative HPLC column (22 × 150 mm). The column was eluted with a gradient of 10–40% of 0.9% trifluoroacetic acid/CH3CN. The final product was lyophilized to a white powder. The identity was verified by mass spectroscopy (m/z = 689 and 345, for the di-cation). For diacetyl-d-tubocurarine (7′,12′-diacetoxy-6,6′-dimethoxy-2,2′,2′-trimethyltubocuraranium dinitrate), 1.07 g of dTC was treated with 2.5 ml of acetic anhydride in 8 ml of glacial acetic acid and 874 mg of sodium acetate, essentially according to Dutcher (23Dutcher J.D. J. Am. Chem. Soc. 1952; 74: 2221-2225Crossref Scopus (14) Google Scholar). The product was isolated by crystallization of the nitrate salt with a yield of 66%. This product was 95% pure as judged by reversed phase HPLC; the remaining contaminant corresponded to a monoacetylated compound. For iodo-chondocurarine (7′,12′-dihydroxy-6,6′-dimethoxy-13′-iodo-2,2,2′,2′-tetramethyltubocuraranium ditrifluoroacetate), ICl (0.5 mmol) in MeOH was slowly added to 0.2 mmol of chondocurarine (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) dissolved in 0.1 m acetate buffer (pH 5.6, 100 ml) on ice. After 30 min of reaction, 0.5 mmol of neat 2-mercaptoethanol was added to quench the excess ICl. The mixture was diluted to 250 ml, adjusted to pH 8.0 using NH4OH, and subjected to a CM-25 Sephadex cation exchange column chromatography. The column was eluted with a linear gradient from 50 to 300 mm NH4HCO3, pH 8.0. The fractions containing pure product were identified by reversed phase HPLC, pooled, and lyophilized. The dry salts were dissolved in 0.1% trifluoroacetic acid/H2O and applied to a 21.2 × 150-mm Ultraprep C18 reversed phase HPLC column (Beckman) in four batches and then eluted with a gradient of 20–50% CH3CN over 60 min. The pure fractions were pooled and lyophilized to a white powder (47.4 mg, 49 μmol, 25%). This product was judged pure by HPLC, UV-visible spectroscopy, 1H NMR spectroscopy (two aromatic protons were shifted to δ7.62 and δ7.12 compared with δ7.11 and δ7.06 of chondocurarine), and mass spectrometry (m/z = 749.2, m/z = 375.1 for the double ion). For di-demethyl-d-tubocurarine (6,6′,7′,12′-tetrahydroxy-2,2′,2′-trimethyltubocuraranium ditrifluoroacetate), dTC (10 mg) was heated to 160 °C in 300 μl of phosphoric acid. The reaction mixture was diluted with 0.1% trifluoroacetic acid/H2O and applied to a preparative reversed phase HPLC column. A major and a minor product were isolated, both essentially pure, and lyophilized. The major product corresponded to 6,6′-didemethyl-d-tubocurarine by mass spectroscopy (m/z = 581.2 and m/z = 291.2 for the double ion). The minor product was singly demethylated. For the expression studies, the cDNA of each subunit of the mouse muscle AChR (α, β, γ, and δ) was released from the original plasmid by using appropriate combinations of restriction enzymes and purified by agarose gel electrophoresis. The cDNA for each of the four subunits was subsequently cloned into the eukaryotic expression vector pCDNA3 (Invitrogen) to create the expression plasmids pCDNA3.NA-α, pCDNA3.NA-β, pCDNA3.NA-δ, and pCDNA3.NA-γ, respectively. The pCDNA3.NA-γ plasmid was digested with the restriction enzymes EcoRV andXhoI, and the resulting two DNA fragments were purified by agarose gel electrophoresis. The small DNA fragment (NA-γ.S) contained the sites for introducing the desired mutations, and the large DNA fragment (pCDNA.NA-γ.L) contained the remainder of the γ-subunit cDNA attached to the pCDNA vector; it was saved for later use. The NA-γ.S DNA fragment was subsequently cloned into the pBlueScript vector (Stratagene), which was pre-digested with the restriction enzymes EcoRV and XhoI. Point mutations were created by site-directed mutagenesis using appropriate pairs of mutation-specific oligonucleotide primers and the QuickChange mutagenesis kit, following the manufacturer's protocol. Subsequently, the plasmid DNA was transformed into the Escherichia coliXL-1 blue. Overnight cultures of three to four colonies from each transformation plate were grown, and plasmids were purified from these cultures using the Wizard plasmid purification kit (Promega, Madison, WI). The sequence of the entire DNA insert of each putative mutant plasmid was analyzed to confirm the desired mutation and to verify the sequence fidelity of the rest of the DNA. The NA-γ.S DNA fragment containing a given mutation was then released from the plasmid using the restriction enzymes EcoRV andXhoI, purified by preparative agarose gel electrophoresis, and ligated with the previously purified pCDNA.NA-γ.L DNA fragment (see above). After verification by DNA sequencing, a recombinant plasmid containing the mutated full-length NA-γ cDNA was selected for large scale purification. Large scale purifications were carried out using either the Endofree Plasmid Mega (for 500 ml of culture) or the Endofree Plasmid Giga (2,500 ml of culture) kit. This purification was necessary for optimal expression of AChRs. HEK293 cells were grown to 70–90% confluence in 100-mm culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 0.1 unit/ml penicillin, and 0.1 μg/ml streptomycin. Generally, cells passaged less than 15 times in culture were used for transient expression of the wild type and the mutant AChRs. For transient expression, the pCDNA3-based AChR subunit expression plasmids were mixed in a ratio of 2:1:1:1 of α/β/γ/δ (21 μg of total DNA/100-mm dish). A stock solution of PEI (25kD, branched form) was prepared at a concentration of 4.3 mg/ml (0.1 m in nitrogen) in phosphate-buffered saline (PBS) (24Densmore C.L. Orson F.M., Xu, B. Kinsey B.M. Waldrep J.C. Hua P. Bhogal B. Knight V. Mol. Ther. 2000; 1: 180-188Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). For each dish, 6.3 μl of PEI stock was diluted with 1.5 ml of sterile PBS, and the DNA was then added and mixed. The PBS/PEI/DNA mixture was incubated for 20–45 min at ambient temperature before addition to 9 ml of culture medium. The old culture medium was then removed from the dish, and the PBS/PEI/DNA mixture was added. Cells were returned to the incubator and harvested 48–72 h after transfection. [3H]Acetylcholine binding and binding of 125I-α-BgTx to Torpedo AChR was carried out as described previously (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). For determination of binding constants to mutant mouse AChR expressed in tissue culture cells, the following protocol was observed. Cells were scraped into PBS, centrifuged, and resuspended in high potassium/Ringer's containing 140 mm KCl, 5.4 mm NaCl, 1.8 mmCaCl2, 1.7 mm MgCl2, 25 mm Hepes, pH 7.0, with 30 μg/ml bovine serum albumin (16Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). Samples were incubated with the indicated concentration of competing ligand for 30 min at ambient temperature before the addition of 0.1–0.3 nm125I-α-BgTx (2000 Ci/mmol). A first order binding rate was observed for more than 2 h. Binding was stopped at 2 h with excess, cold, non-radioactive BgTx, and the samples were stored on ice. Samples were filtered and washed with PBS over GF/C filters that had been soaked cold overnight with 4% Carnation Instant Nonfat Dry Milk in potassium/Ringer's buffer. Filters were counted in a gamma counter (Beckman). Data were plotted and analyzed using SigmaPlot (Jandel, SPSS, Chicago, IL). To block binding to the αδ site, 30–80 nm CTX was added to each assay for most experiments; experiments using wild type receptors confirmed that this concentration of conotoxin only blocks ligand binding to the αδ site and does not change the affinity of the αγ site (25Papineni R.V. Sanchez J.U. Baksi K. Willcockson I.U. Pedersen S.E. J. Biol. Chem. 2001; 276: 23589-23598Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We observed that the mouse AChR expressed in HEK293 cells displayed higher affinity for tubocurine and for iodo-dTC than the affinities observed previously for binding to the mouse AChR expressed by BC3H1 cells (22Papineni R.V. Pedersen S.E. J. Biol. Chem. 1997; 272: 24891-24898Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We found that the lower affinities observed earlier had been due to the higher pH conditions of the former assays (∼7.5–8.0) and also to unidentified components in the former tissue culture media that specifically affected the binding of iodo-dTC. Nonetheless, the affinity changes of tubocurine and iodo-dTC described below were consistent with our earlier conclusions that the 2′-N likely interacted with the α-subunits or with conserved aspects of the heterologous subunits (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 22Papineni R.V. Pedersen S.E. J. Biol. Chem. 1997; 272: 24891-24898Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and that the 12′, 13′ region interacted with the heterologous subunits. For each binding curve, theK I was determined by fitting to the equation for single site binding f =A(K I/(K I +L)) + background, where A represents maximum fmol bound, and L is the ligand concentration. Binding curves obtained in the absence of CTX were fitted to models for two-site binding with either equal or unequal site stoichiometries as described previously (22Papineni R.V. Pedersen S.E. J. Biol. Chem. 1997; 272: 24891-24898Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To determine the degree of interaction between a site on the ligand and a site on the receptor, we used a double mutant thermodynamic cycle analysis (26Ackers G.K. Smith F.R. Annu. Rev. Biochem. 1985; 54: 597-629Crossref PubMed Scopus (197) Google Scholar, 27Pedersen S.E. Lurtz M.M. Papineni R.V. Methods Enzymol. 1999; 294: 117-135Crossref PubMed Scopus (7) Google Scholar). Four different binding curves were obtained in a single experiment; two ligands differing in a discrete position were measured with both wild type and mutant AChR. The interaction factor Ω was then calculated from theK I values using the equation Ω = (K W,L1/K W,L2)/(K m,L1/K M,L2), where the subscripts indicate the following: W for wild type AChR, M for mutant receptor, and L1 andL2 for the two ligands being compared. An Ω value significantly different from 1 indicates an interaction between the functional group on the ligand and the amino acid of the receptor. Energy minimizations of dTC and the AChBP (Ref. 20Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1586) Google Scholar, Protein Data Bank code 1I9B) were conducted with the software package HyperChem (versus 5.1, Hypercube Inc., Gainesville, FL) using the MM+ force field. The initial structure of dTC was taken from the coordinates of a crystal form (28Reynolds C.D. Palmer R.A. Acta Crystallogr. Sect. B Struct. Sci. 1976; 32: 1431-1439Crossref Google Scholar) and was energy-minimized, which produced only minor changes in the structure. Initial simulations were sped up by using a reduced structure of the AChBP. Three of the five subunits were deleted; of the remaining two subunits, only the amino acids in immediate ligand contact plus one layer of surrounding amino acids were considered during energy minimization. For the initial manual alignment of dTC in the AChBP-binding site, dTC was placed in the binding site with the 2′-ammonium at the site of the ethanesulfonic acid-piperazine nitrogen of Hepes, a ligand found in the original structure of the binding site (20Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1586) Google Scholar). dTC was energy-minimized in several rounds without allowing changes in protein structure. To resolve steric conflicts or trapping in local energy minima, occasional manual changes in dTC location were made. When a preliminary structure was obtained for bound dTC, it was then merged with two complete subunits of the acetylcholine-binding protein. Subsequent dTC energy minimization included minimization of all contact residues in the context of two complete AChBP subunits. To mimic the structure of the mouse AChR αγ-binding site, the aligned, homologous mouse residues were substituted for the side chains of contact residues. The substituted side chains are shown in Fig.8 C. After each substitution, the side chain was minimized to conform to the surrounding protein environment. dTC was then energy-minimized in the context of the new structure. Two separate rounds of minimizations were conducted. First, dTC was minimized while the protein was kept rigid. Second, after the approximate dTC conformation was found, dTC and 31 nearby residues were then all minimized collectively to yield the final structure. In order to examine the proximal relationship of AChR amino acid residues to functional groups on dTC, we assessed the degree of interaction by double mutant thermodynamic cycle analysis. In general, the affinities of mutant and wild type AChRs were measured for a pair of dTC analogs. The four K I values thus obtained were used to determine the value of the interaction coefficient, Ω (see “Experimental Procedures”). An initial goal was to identify amino acids that had specific interactions with dTC. Our previous work (21Pedersen S.E. Papineni R.V. J. Biol. Chem. 1995; 270: 31141-31150Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 22Papineni R.V. Pedersen S.E. J. Biol. Chem. 1997; 272: 24891-24898Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) had shown that modification of dTC to metocurine or to iodo-dTC shifted the ligand affinities and that the magnitude of the shifts differed at the two binding sites. Comparison of dTC with these analogs would test for interactions at the 2-N ammonium and at the 7′-, 12′-, and 13′-C positions. Therefore, our preliminary experiments utilized these two analogs, although not exclusively. To determine amino acids likely to interact at these dTC sites, we focused on those γ-subunit residues that had been identified to contribute to site-selective binding. Because dTC binds with higher affinity to the αγ site than the αδ site on bothTorpedo AChR and mouse AChR, γ-subunit amino acids were initially mutated to those amino acids found in the homologous, aligned positions of δ- or ε-subunits of either Torpedo or mouse muscle AChR. We first examined those residues identified by chimeric constructs to contribute to site-selective dTC binding: γIle116, γTyr117, and γSer161(16Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). Conservative mutations of γI116M and γI116V displayed small, 2–4-fold affinity increases for metocurine over the wild type AChR and small affinity decreases for" @default.
• W2024489413 created "2016-06-24" @default.
• W2024489413 creator A5000821682 @default.
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• W2024489413 date "2002-11-01" @default.
• W2024489413 modified "2023-10-17" @default.
• W2024489413 title "Orientation of d-Tubocurarine in the Muscle Nicotinic Acetylcholine Receptor-binding Site" @default.
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