Lysine Scanning Mutagenesis Delineates Structural Model of the Nicotinic Receptor Ligand Binding Domain
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m203396200
ISSN1083-351X
AutoresSteven M. Sine, Hailong Wang, Nina Bren,
Tópico(s)Insect and Pesticide Research
ResumoNicotinic acetylcholine receptors (AChR) and their relatives mediate rapid chemical transmission throughout the nervous system, yet their atomic structures remain elusive. Here we use lysine scanning mutagenesis to determine the orientation of residue side chains toward core hydrophobic or surface hydrophilic environments and use this information to build a structural model of the ligand binding region of the AChR from adult human muscle. The resulting side-chain orientations allow assignment of residue equivalence between AChR subunits and an acetylcholine binding protein solved by x-ray crystallography, providing the foundation for homology modeling. The resulting structural model of the AChR provides a picture of the ACh binding site and predicts novel pairs of residues that stabilize subunit interfaces. The overall results suggest that lysine scanning can provide the basis for structural modeling of other members of the AChR superfamily as well as of other proteins with repeating structures delimiting a hydrophobic core. Nicotinic acetylcholine receptors (AChR) and their relatives mediate rapid chemical transmission throughout the nervous system, yet their atomic structures remain elusive. Here we use lysine scanning mutagenesis to determine the orientation of residue side chains toward core hydrophobic or surface hydrophilic environments and use this information to build a structural model of the ligand binding region of the AChR from adult human muscle. The resulting side-chain orientations allow assignment of residue equivalence between AChR subunits and an acetylcholine binding protein solved by x-ray crystallography, providing the foundation for homology modeling. The resulting structural model of the AChR provides a picture of the ACh binding site and predicts novel pairs of residues that stabilize subunit interfaces. The overall results suggest that lysine scanning can provide the basis for structural modeling of other members of the AChR superfamily as well as of other proteins with repeating structures delimiting a hydrophobic core. acetylcholine receptor acetylcholine binding protein α-bungarotoxin α-conotoxin GI Traditional methods for atomic structural determination use x-ray crystallography or NMR spectroscopy. However, many proteins do not form crystals, and many are too large to solve by NMR. New methods are therefore urgently needed to determine structures of such intractable proteins. Here we develop a mutagenesis-based modeling method and apply it to the ligand binding region of the nicotinic AChR1 from adult human muscle. The method uses lysine scanning to distinguish core hydrophobic from surface hydrophilic orientations of residue side chains and uses this information to align residues in AChR subunits with equivalent residues in the homologous AChBP, which was solved by x-ray crystallography (1Brejc K. van Dijk W. Klassen R. Schuurmans M. van der Oost J. Smit A. Sixma T. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1580) Google Scholar). The experimentally determined alignment forms the foundation for generating an atomic structural model of the ligand binding region of the heteromeric AChR. Knowledge of nicotinic receptor structure advanced along two independent lines of investigation over the past decade. The first line stemmed from primary sequence data deduced from cloning AChR subunits and their relatives (2Noda M. Takahashi H. Tanabe T. Toyosato M. Furutani Y. Hirose T. Asai M. Inayama S. Miyata T. Numa S. Nature. 1982; 299: 793-797Crossref PubMed Scopus (503) Google Scholar). These studies included prediction of membrane spanning regions using hydropathy analysis, prediction of secondary structure from the sequence data (3Le Novère N. Corringer P.-J. Changeux J.-P. Biophys. J. 1999; 76: 2329-2345Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), identification of key residues by affinity labeling together with microsequencing, and site-directed mutagenesis combined with functional measurements (4Prince R. Sine S.M. Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Landes Bioscience, Austin, TX1997: 31-59Google Scholar, 5Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (564) Google Scholar, 6Corringer P.J. Le Novère Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar). The emerging picture of the AChR indicated a pentamer in which about half of each subunit contributes to an N-terminal extracellular domain that harbors the ACh binding sites. The remainder of each subunit consists of four transmembrane domains, the second of which contributes to the ion channel, and a large cytoplasmic domain between the third and fourth transmembrane domains. Mutagenesis and site-directed labeling localized the ACh binding sites to interfaces between α and non-α subunits (4Prince R. Sine S.M. Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Landes Bioscience, Austin, TX1997: 31-59Google Scholar, 6Corringer P.J. Le Novère Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar), whereas studies of residue accessibility and residues affecting ion permeability revealed the channel gate and ion selectivity filter within the second transmembrane domain (7Wilson G. Karlin A. Neuron. 1998; 20: 1269-1281Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 8Corringer P.J. Bertrand S. Galzi J.L. Devillers-Thiery A. Changeux J.P. Bertrand D. Neuron. 1999; 22: 831-843Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Advances along the second line of investigation came from cryo-electron microscopy of two-dimensional arrays of AChRs from Torpedo. Data at 9-Å resolution revealed the overall shape and dimensions of the AChR and identified secondary structural elements in the vicinity of the ACh binding site and the ion channel (9Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (717) Google Scholar). The most recent data at 4.6-Å resolution revealed several aligned β strands near the putative ACh binding site, and a fenestrated basket-like structure extending into the cytoplasm (10Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar). Images obtained following rapid application of ACh revealed that each subunit twists about the axis normal to the membrane when the ion channel opens (11Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (909) Google Scholar). Overall, the cryo-electron microscopy data could be reconciled with the mutagenesis, labeling, and functional measurements, although the precise locations of the ACh binding sites and channel gate remained controversial. Atomic structural insight recently emerged from the crystal structure of AChBP, a 120-kDa acetylcholine binding protein homologous to the ligand binding region of the AChR (1Brejc K. van Dijk W. Klassen R. Schuurmans M. van der Oost J. Smit A. Sixma T. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1580) Google Scholar). The sequence of AChBP is 23.9% identical to that of the homomeric α7 AChR and harbors residues considered diagnostic of nicotinic receptor α subunits, including disulfide-bonded cysteines and aromatic residues that contribute to the ACh binding site. These conserved elements suggest that AChBP may provide a model for the three-dimensional structure of the ligand binding domain of the AChR and its relatives. Here we combine lysine scanning mutagenesis, ligand binding measurements, and homology modeling to deduce a structural model of the ligand binding region of the nicotinic AChR from adult human muscle. Lysine scanning of the ε subunit reveals that it oligomerizes with complementary AChR subunits when lysine is placed at alternating positions along the protein chain, indicating the presence of β strands and establishing orientation of the side chains toward core hydrophobic and surface hydrophilic environments. The side-chain orientations allow alignment of equivalent residues between the ε subunit and AChBP, forming the foundation for homology modeling. The resulting atomic structural model provides a detailed picture of the AChR ligand binding site and discloses novel residue pairs that stabilize subunit interfaces. 125I-Labeled α-bgt was from PerkinElmer Life Sciences, d-tubocurarine chloride from ICN Pharmaceuticals, Inc., 293 human embryonic kidney cells (293 HEK) were from the American Type Culture Collection, α-conotoxin GI was from Sigma Chemical Co., and the fully methylated analog ofd-tubocurarine, metocurine iodide, was a gift from the Eli Lilly Co. Human adult AChR subunit cDNAs were obtained as described previously (12Ohno K. Wang H.-L. Milone M. Bren N. Brengman J.M. Nakano S. Quiram P. Pruitt J.N. Sine S.M. Engel A.G. Neuron. 1996; 17: 157-170Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and subcloned into the cytomegalovirus-based expression vector, pRBG4, as described (13Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). Mutations were constructed using the QuikChange kit from Stratagene. All mutations were confirmed by dideoxy sequencing. HEK cells were transfected with mutant or wild type AChR subunit cDNAs using calcium phosphate precipitation as described (13Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). In all experiments, AChR subunit cDNAs were co-transfected in the following quantities per 10-cm plate of HEK cells: α (13.6 μg) and β, and ε and δ (6.8 μg). Three days after transfection, intact HEK cells were harvested by gentle agitation in phosphate-buffered saline plus 5 mm EDTA. Ligand binding to intact cells was measured by competition against the initial rate of125I-α-bungarotoxin (α-bgt) binding (14Sine 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 high potassium Ringer's solution, and divided into aliquots for ligand binding measurements. Potassium Ringer's solution contains 140 mm KCl, 5.4 mm NaCl, 1.8 mmCaCl2, 1.7 mm MgCl2, 25 mm HEPES, 30 mg/liter bovine serum albumin, adjusted to pH 7.4 with 10–11 mm NaOH. Specified concentrations of competing ligand were added 30 min prior to adding125I-α-bgt, which was allowed to bind for 30 min to occupy approximately half of the surface receptors. Binding was terminated by addition of 2 ml of potassium Ringer's solution containing 100 μm d-tubocurarine chloride. Cells were immediately filtered through Whatman GF-B filters using a Brandel cell harvester and washed five times with 2 ml of potassium Ringer's solution. Prior to use, filters were soaked in water containing 4% dried skim milk for at least 2 h. Nonspecific binding was determined in the presence of 1 mm d-tubocurarine. The total number of α-bgt binding sites was determined by incubation with the toxin for 90 min. The initial rate of α-bgt binding was calculated as described (14Sine S.M. Taylor P. J. Biol. Chem. 1979; 254: 3315-3325Abstract Full Text PDF PubMed Google Scholar) to yield fractional ligand occupancy. Competition measurements were analyzed according to one of the following equations: the Hill equation (Equation 1), the sum of two binding sites present in equal numbers (Equation 2), one binding site plus a constant (Equation 3), the sum of two binding sites present in unequal numbers (Equation 4), and weighted contributions of Equations 2 and 3 (Equation 5), 1−Y=1/(1+([ligand]/KApp)n)Equation 1 1−Y=0.5/(1+[ligand]/KA)+0.5/(1+[ligand]/KB)Equation 2 1−Y=FractA[1/(1+[ligand]/KA)]+1−FractAEquation 3 1−Y=FractA[1/(1+[ligand]/KA)]+(1−FractA)(1/(1+[ligand]/KB)Equation 4 1−Y=Fract2site[1/(1+[ligand]/KA)+1/(1+[ligand]/KB)]+(1−Fract2site)[FractA/(1+[ligand]/KA)+1−FractA]Equation 5 where Y is fractional ligand occupancy, nis the Hill coefficient, K APP is an apparent dissociation constant, K A andK B are intrinsic dissociation constants, FractA is the fraction of sites with dissociation constantK A, and Fract2-site is the fraction of binding sites with equal numbers of K A andK B sites. We generated a homology model of the major extracellular domain of the adult human AChR using version 6.0 of the program MODELER (15Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10563) Google Scholar), together with spatial restraints provided by the AChBP structure (1Brejc K. van Dijk W. Klassen R. Schuurmans M. van der Oost J. Smit A. Sixma T. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1580) Google Scholar). We aligned AChR and AChBP sequences based on side-chain dispositions determined by lysine scanning combined with ligand binding measurements, as described under "Results" (Table Iand Fig. 4). However, to model strand β1, spatial restraints were removed for AChR εL40 and two flanking residues to account for its surface exposure (see Fig. 5), which is contrary to the corresponding aligned residue, Ile-38, in AChBP. To maintain complementarity between subunits at their interfaces, all five subunits were modeled simultaneously. AChR α subunits were matched to A and C subunits of AChBP, and the ε, δ, and β subunits were matched with the B, D, and E subunits, respectively. We used the "patch" command in MODELER to constrain coordinates of cysteines 128 and 142, which form a disulfide bond in each subunit. Among several options in MODELER, we selected the "refine1" mode, which generates the highest level of refinement using conjugate gradients coupled with simulated annealing and molecular dynamics. Modeling included all polar hydrogens to allow for main-chain hydrogen bonding but omitted non-polar hydrogens. We used MODELER to generate 100 different structures, and evaluated each structure using the programs PROCHECK (34Laskowski R. MacArthur M. Moss D. Thornton J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and PROFILES-3D (InsightII, Accelrys Inc., San Diego, CA). Among the 100 structures, ∼10 were of high quality and very similar, and we selected the best of these for further modeling.Table IExpression and CTx GI competition parameters for lysine mutationsAChRεAChBPExpressionK AK BFraction Aα2βεδ1.01.9E−91.1E−70.5α2βδ20.35.8E−9NC1-aNC, a second component was present but that there was no competition at CTx GI concentrations greater than 30 μm (see Fig. 1 b). For AChBP, residues highlighted in boldface orient toward the hydrophobic core (see Fig. 4, legend).0.52Strand β1T28KPro-260.682.7E−95.2E−80.5V29KVal-270.224.2E−9NC0.55T30KAla-281.12.2E−98.7E−80.5I31KVal-290.195.3E−9NC0.55S32KSer-301.081.3E−92.6E−80.5L33KVal-310.196.8E−9NC0.57K34Ser-32NM1-bNM, residue was not mutated.V35KLeu-330.198.1E−9NC0.59T36KLys-340.581.8E−91.7E−80.5L37KPhe-350.173.8E−9NC0.54T38KIle-361.191.1E−91.2E−80.5N39KAsn-370.422.6E−95.6E−90.5L40KIle-380.612.4E−93.2E−80.5I41KLeu-390.71.1E−91.6E−80.5S42KGlu-4011.6E−93.5E−80.5L43KVal-410.393.3E−9NC0.56N44KAsn-420.912.3E−91.5E−70.5Strand β2T49KGlu-470.921.0E−95.8E−80.5L50KVal-480.183.2E−9NC0.58T51KAsp-490.793.9E−93.6E−80.5T52KVal-500.173.6E−9NC0.49S53KVal-510.691.3E−91.4E−80.5V54KPhe-520.193.2E−9NC0.48W55KTrp-530.662.3E−91.3E−80.5I56KGln-540.252.0E−8NC0.7G57KGln-550.698.3E−98.3E−90.5I58KThr-560.211.8E−8NC0.62D59KThr-570.771.4E−92.3E−70.5W60KTrp-580.153.4E−9NC0.58Q61KSer-591.017.70E−108.1E−80.5β2–3 linkerI75KPro-710.273.0E−9NC0.51E76KAsp-720.832.0E−92.0E−70.5Strand β3T77KGln-730.814.0E−96.3E−70.5L78KVal-740.236.0E−9NC0.53R79Ser-75NMV80KVal-760.227.9E−9NC0.43Strand β4I90KLeu-860.221.7E−9NC0.48V91KAla-870.75.6E−99.3E−90.5L92KAla-880.371.3E−8NC0.79E93KTyr-890.794.1E−91.1E−80.5Strand β5G101KGlu-960.983.0E−92.0E−75.0E−1V102KVal-9714.1E−93.5E−75.0E−1β5–5′ linkerA103KLeu-981.013.6E−91.99E−75.0E−1Y104KThr-990.951.5E−91.9E−85.0E−1D105KPro-1000.892.5E−99.9E−80.5A106KGln-1010.391.5E−91.3E−80.5Strand β5′N107KLeu-1020.813.3E−92.8E−80.5V108KAla-1030.28.1E−9NC0.57L109KArg-1040.773.7E−9NC0.7V110KVal-1050.186.4E−9NC0.49Y111KVal-1060.793.1E−9NC0.62β5′–6 linkerE112KSer-0170.751.1E−92.6E−80.5G113KAsp-1080.671.1E−95.3E−80.5G114KGly-1090.124.9E−9NC0.52Strand β6S115KGlu-1100.971.4E−97.6E−80.5V116KVal-1110.095.0E−9NC0.55T117KLeu-1120.943.5E−9NC0.59W118KTyr-1130.176.4E−9NC0.71L119KMet-1140.483.8E−93.8E−90.5P120KPro-1150.951.5E−91.4E−80.5P121KSer-1161.054.7E−9NC0.5A122KIle-1171.111.4E−91.95E−80.5I123KArg-1180.729.00E−102.1E−80.5Y124KGln-1190.548.60E−101.5E−80.5R125Arg-120NMS126KPhe-1210.181.9E−8NC0.62V127KSer-1220.91.4E−92.1E−80.5C128Cys-123NMStrand β7N141Thr-135NMC142Cys-136NMS143Arg-137NML144KIle-1380.181.2E−8NC0.52I145KLys-1390.653.2E−91.4E−80.5F146KIle-1400.173.0E−9NC0.43R147Gly-141NMS148KSer-1420.261.2E−8NC0.66Strand β8E155KGlu-1490.511.4E−91.8E−80.5V156KIle-1500.221.2E−8NC0.54E157KSer-1510.952.7E−92.4E−70.5F158KVal-1520.27.0E−9NC0.44T159KAsp-1531.042.6E−91.8E−80.5β8–9 linkerF160KPro-1540.291.5E−8NC0.68A161KThr-1550.286.5E−9NC0.55V162KThr-15612.7E−92.2E−70.5D163KGlu-1570.634.5E−94.5E−50.81I172KAsn-1580.22.0E−9NC0.5D173KSer-1590.942.3E−97.0E−80.5I174KAsp-1600.311.4E−91.9E−70.5D175KAsp-1610.724.2E−91.3E−60.5T176KSer-1620.962.2E−94.0E−80.5E177KGlu-1630.913.8E−92.1E−70.5A178KTyr-1641.082.4E−95.9E−80.5Y179KPhe-1650.542.5E−91.8E−70.5T180KSer-1660.682.8E−93.4E−80.5E181KGln-1670.344.6E−9NC0.54N182KTyr-1680.493.0E−98.5E−80.5Strand β9E184KArg-1700.811.9E−92.2E−80.5W185KPhe-1710.235.4E−9NC0.51A186KGlu-17212.9E−92.6E−80.5I187KIle-1730.257.6E−9NC0.51D188KLeu-1740.772.1E−92.4E−80.5F189KAsp-1750.711.5E−92.2E−80.5C190KVal-1760.239.0E−9NC0.58P191KThr-1770.527.8E−91.2E−50.68G192KGln-1780.272.3E−9NC0.5V193KLys-1791.032.8E−91.3E−70.5I194KLys-1800.392.3E−8NC0.89R195Asn-181NMStrand β10E207KTyr-1920.913.7E−91.3E−80.5T208KGlu-1930.431.7E−8NC0.86D209KAsp-1940.762.4E−92.7E−80.5V210KVal-1950.237.4E−9NC0.54I211KGlu-1960.872.4E−94.3E−80.5Y212KVal-1970.237.2E−9NC0.58S213KSer-1980.72.2E−94.2E−80.5L214KLeu-1990.185.2E−9NC0.53I215KAsn-2000.346.6E−9NC0.76I216KPhe-2010.232.7E−9NC0.51R217Arg-202NMExpression is the total number of cell surface α-bgt binding sites relative to that for wild type α2βεδ AChRs (see Fig.1 a and "Experimental Procedures"). For receptors in which CTx GI competed against all α-bgt binding (Fig. 1 b),K A and K B are dissociation constants determined by fitting Equation 2 to the data. For receptors in which Ctx GI did not compete against all α-bgt binding (highlighted in boldface; see Fig.1 b), K A is the dissociation for the high affinity component, and Fraction A is the fraction of sites with dissociation constant K A determined by fitting Equation 3 to the data.1-a NC, a second component was present but that there was no competition at CTx GI concentrations greater than 30 μm (see Fig. 1 b). For AChBP, residues highlighted in boldface orient toward the hydrophobic core (see Fig. 4, legend).1-b NM, residue was not mutated. Open table in a new tab Figure 5Comparison of side-chain dispositions of εL40 and AChBP Ile-38 and local structures of the main chains of strand β1. Strands of the ε subunit are colored yellow, and AChBP is coloredgreen. Note εL40 projects on the protein surface and allows smooth bending of the main chain and co-alignment of strands β1 and β2, whereas Ile-38 projects into the hydrophobic core and coincides with a kink in the main chain of strand β1.View Large Image Figure ViewerDownload (PPT) Expression is the total number of cell surface α-bgt binding sites relative to that for wild type α2βεδ AChRs (see Fig.1 a and "Experimental Procedures"). For receptors in which CTx GI competed against all α-bgt binding (Fig. 1 b),K A and K B are dissociation constants determined by fitting Equation 2 to the data. For receptors in which Ctx GI did not compete against all α-bgt binding (highlighted in boldface; see Fig.1 b), K A is the dissociation for the high affinity component, and Fraction A is the fraction of sites with dissociation constant K A determined by fitting Equation 3 to the data. The major insertions requiring additional modeling were the linkers between strands β8 and β9 in β, ε, and δ subunits, which contain eight, eight, and eleven inserted residues, respectively. The α subunit contains only one insertion in this linker, which MODELER is designed to accommodate (17Fiser A. Sanchez R. Melo F. Sali A. Watanabe M. Roux R. MacKerell A. Becker O. Computational Biochemistry and Biophysics. Marcel Dekker Inc., New York2000: 275-312Google Scholar). To model the β8–β9 linker in the ε subunit, a second round of MODELER was employed using different sequence alignments in this region and constraining coordinates of all atoms except those of the β8–β9 linker. This was an empirical process, and we selected structures that satisfied several criteria. First, we required εF160 in this linker, and equivalent residues in other subunits, to project into the hydrophobic core to account for the observation that εF160K produced predominantly misfolded subunits (Table II). Second, we selected structures in which endogenous positively charged residues in the linker project away from the hydrophobic interior; these included εK171, εK167, δK164, δK167, and δR170. Third, we required εD175 and δD180 to approach within 10–15 Å of αC192/193 determined in cross-linking experiments (16Czajkowski C. Karlin A. J. Biol. Chem. 1995; 270: 3160-3164Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and within 15–20 Å of εL119 or δL121 determined from double-mutant cycles analysis of α-neurotoxin binding (18Malany S. Osaka H. Sine S.M. Taylor P. Biochemistry. 2000; 39: 15388-15398Crossref PubMed Scopus (32) Google Scholar). Although these constraints are qualitative, in practice they eliminated many candidate structures. PROCHECK and PROFILES 3-D were used to select the best structure for further modeling.Table IICtx GI and metocurine competition parameters for lysine mutations yielding subunit-containing (α2βεδ) and subunit-omitted receptors (α2βδ2)MutantCTx GI parametersMetocurine parametersK AK BFraction two-siteα2βδ2/α2βεδK AK BFractionK Aα2βδ2/α2βεδα2βεδ1.9E−91.1E−71.00.07.2E−86.0E−60.50.0I56K7.4E−95.7E−70.540.851.5E−79.5E−60.251.0L92K4.1E−93.1E−70.720.397.9E−76.0E−60.40.25W118K2.1E−91.5E−70.461.24.0E−71.7E−50.330.52S148K6.7E−91.6E−70.320.476.7E−87.5E−60.261.1F160K1.3E−85.5E−80.380.614.2E−84.8E−60.40.25I215K5.7E−93.1E−70.640.341.8E−75.9E−60.220.79P191K2.4E−91.5E−70.900.114.2E−84.8E−60.40.25I194K5.0E−91.8E−70.860.166.9E−89.6E−60.490.02T208K4.9E−91.5E−70.800.258.2E−81.2E−50.450.11For CTx GI competition results, parameters are fits of Equation 5 to the data where K A and K B are dissociation constants and Fraction two-site is the fraction of sites corresponding to α2βεδ receptors, which is used to compute the ratio α2βδ2/α2βεδ. For metocurine competition results, parameters are fits of Equation 4to the data where K A and K B are dissociation constants and Fraction K A is the fraction of sites with dissociation constant K A. The fraction of α2βεδ receptors is computed as twice Fraction A and used to compute the ratio α2βδ2/α2βεδ. Open table in a new tab For CTx GI competition results, parameters are fits of Equation 5 to the data where K A and K B are dissociation constants and Fraction two-site is the fraction of sites corresponding to α2βεδ receptors, which is used to compute the ratio α2βδ2/α2βεδ. For metocurine competition results, parameters are fits of Equation 4to the data where K A and K B are dissociation constants and Fraction K A is the fraction of sites with dissociation constant K A. The fraction of α2βεδ receptors is computed as twice Fraction A and used to compute the ratio α2βδ2/α2βεδ. Finally, we applied two rounds of energy minimization using the program CHARMM (19Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M.J. Comp. Chem. 1983; 4: 187-217Crossref Scopus (13967) Google Scholar), version 27b4. The first round constrained the coordinates of all heavy atoms while allowing movement of hydrogens. The second round constrained the protein main chain while allowing movement of side chains. The final structure was selected based on evaluations using PROCHECK and PROFILES 3-D. We mutated individual residues to lysine along a putative β strand from positions 49 through 61 of the ε subunit from adult human muscle AChR. Each lysine mutation was co-transfected with complementary α, β, and δ subunit cDNAs in 293 HEK cells, followed by quantitation of cell surface receptors using the AChR-specific ligand 125I-α-bgt. The results reveal a pattern of high expression alternating with low expression as lysine is advanced along the protein chain (Fig.1 a). High expression for odd numbered residues approaches that for wild type AChR, whereas low expression for even numbered residues approaches that for subunit-omitted α2βδ2 receptors. High expression likely results from mutant ε subunits that fold and incorporate into pentameric AChRs, whereas low expression likely results from misfolding of the mutant ε subunits, leading to subunit-omitted α2βδ2 receptors (12Ohno K. Wang H.-L. Milone M. Bren N. Brengman J.M. Nakano S. Quiram P. Pruitt J.N. Sine S.M. Engel A.G. Neuron. 1996; 17: 157-170Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 20Sine S.M. Claudio T. J. Biol. Chem. 1991; 266: 19369-19377Abstract Full Text PDF PubMed Google Scholar,21Quiram P. Ohno K. Milone M. Patterson M. Pruitt N. Brengman J. Sine S. Engel A.G. J. Clin. Invest. 1999; 104: 1403-1410Crossref PubMed Scopus (65) Google Scholar). These results provide strong evidence that residues 49 through 61 in the AChR ε subunit form a β strand. Alternatively, lysine mutations at even numbered positions may produce a subunit that can assemble with complementary subunits, but the mutations reduce the efficiency of receptor assembly. To distinguish between omission of the ε subunit and inefficient assembly, we took advantage of the observation that ligand binding properties of the receptor depend on subunit composition (12Ohno K. Wang H.-L. Milone M. Bren N. Brengman J.M. Nakano S. Quiram P. Pruitt J.N. Sine S.M. Engel A.G. Neuron. 1996; 17: 157-170Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 20Sine S.M. Claudio T. J. Biol. Chem. 1991; 266: 19369-19377Abstract Full Text PDF PubMed Google Scholar, 22Blount P. Merlie J.P. Neuron. 1989; 3: 349-357Abstract Full Text PDF PubMed Scopus (228) Google Scholar). Thus for each mutation, we measured binding of ACh and α-conotoxin GI (CTx GI) by competition against the initial rate of 125I-α-bgt binding. Receptors containing the ε subunit (α2βεδ) show a monophasic competition curve for the agonist ACh, whereas receptors lacking the ε subunit (α2βδ2) show a distinctive biphasic curve with a plateau at half occupancy extending over three orders of magnitude in ACh concentration (12Ohno K. Wang H.-L. Milone M. Bren N. Brengman J.M. Nakano S. Quiram P. Pruitt J.N. Sine S.M. Engel A.G. Neuron. 1996; 17: 157-170Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 21Quiram P. Ohno K. Milone M. Patterson M. Pruitt N. Brengman J. Sine S. Engel A.G. J. Clin. Invest. 1999; 104: 1403-1410Crossref PubMed Scopus (65) Google Scholar) (Fig. 1 b). Analogous results are obtained with the competitive antagonist CTx GI, where α2βεδ receptors show a competition curve with two closely spaced components, whereas subunit-omitted receptors show a high affinity component accompanied by a plateau extending over nearly three orders of magnitude of CTx GI concentration. Thus competition profiles for ACh and CTx GI clearly distinguish receptors containing the ε subunit from those with the composition α2βδ2. For the odd numbered lysine mutations, both ACh and CTx GI fully compete against binding of the reporter ligand 125I-α-bgt (Fig. 1 b). Quantitative differences in the competition profiles are observed among the various mutations, because residues in this region contribute to the ligand binding site (23Bren N. Sine S. J. Biol. Chem. 1997; 272: 30793-30798Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 24Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). By contrast, lysine mutations at even numbered positions lead to biphasic profiles characteristic of subunit-omitted α2βδ2 receptors (Fig. 1 b). Together with the alternating pattern of high and low expression, these results indicate that lysine mutations at odd numbered positions permit incorporation of ε subunits into pentameric AChRs, whereas lysine mutations at even numbered positions lead to ε subunits unable to incorporate into pentamers. For even numbered lysine mutations in strand β2, all but one show a plateau in the competition curve corresponding to ∼50% of the binding sites, as observed for subunit-omitted α2βδ2 receptors. However, for the εI56K mutation, the plateau corresponded to 28% of the binding sites (Fig.1 b). Because a plateau of 50% corresponds to 100% α2βδ2 receptors, a plateau of 28% indicates 56% α2βδ2 and 44% containing the mutant ε subunit (i.e. α2βδεI56K). Co-expression of the εI56K mutant yields total expression of α-bgt binding sites of 25% of control, of which only 11% contain the mutant ε subunit. The reduced expression, together with the reduced plateau in the competition measurements, mirrors observations for a disease-causing mutation in the human ε subunit (12Ohno K. Wang H.-L. Milone M. Bren N. Brengman J.M. Nakano S. Quiram P. Pruitt J.N. Sine S.M. Engel A.G. Neuron. 1996; 17: 157-170Abstract Full Text Full Text PD
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