Lysine Scanning Mutagenesis Delineates Structural Model of the Nicotinic Receptor Ligand Binding Domain

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. 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 the and ion the second transmembrane domain A. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. S. A. Changeux J.P. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). along the second line of investigation from of of from the overall and of the AChR and secondary structural in the of the ACh binding site and the ion N. J. PubMed Scopus Google Scholar). The data the ACh binding and a structure the A. Y. M. N. J. 1999; PubMed Scopus Google Scholar). rapid of ACh that each subunit about the to the membrane the ion N. Nature. 1995; PubMed Scopus Google Scholar). the data with the and functional the of the ACh binding sites and structural from the structure of AChBP, a 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 to that of the AChR and harbors residues of nicotinic receptor α and residues that to the ACh binding These suggest that provide a model for the structure of the ligand binding domain of the AChR and Here we lysine scanning ligand binding and homology modeling to a structural model of the ligand binding region of the nicotinic AChR from adult human muscle. scanning of the subunit that it with AChR subunits lysine along the protein the of and orientation of the side chains toward core hydrophobic and surface hydrophilic The side-chain orientations allow alignment of equivalent residues between the subunit and AChBP, the foundation for homology modeling. The resulting atomic structural model provides a picture of the AChR ligand binding site and novel residue pairs that stabilize subunit interfaces. was from from human from the α-conotoxin GI was from and the was a from the adult AChR subunit as K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google and the as S.M. S. A. PubMed Scopus Google Scholar). using the from by with or AChR subunit using as S.M. S. A. PubMed Scopus Google Scholar). AChR subunit in the of α and and and by in binding to was by the binding S.M. J. Full Text PDF PubMed Google Scholar). the in and for ligand binding to with of ligand to which was to for to half of the surface was by of of using a and with of to in for binding was determined in the of The of binding sites was determined by with the for The of binding was as S.M. J. Full Text PDF PubMed Google to ligand measurements to of the the the of two binding sites in binding site a the of two binding sites in and of and ligand the an are the of sites with and the of binding sites with of sites. a homology model of the extracellular domain of the adult human AChR using of the A. J. PubMed Scopus Google Scholar), together with by the 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). AChR and side-chain determined by lysine scanning combined with ligand binding as However, to model for AChR and two residues to for surface which to the in between subunits their subunits AChR α subunits to and subunits of AChBP, and the and subunits with the and the in to of and which form a in each in we the which the of using with and included to allow for to and each structure using the R. M. J. Google and the of and and we the of for and GI for lysine a second was that was GI AChBP, residues in toward the hydrophobic core residue was not the of surface binding sites to that for a and receptors in which GI binding and are determined by to the receptors in which GI not binding in the for the affinity and the of sites with determined by to the a second was that was GI AChBP, residues in toward the hydrophobic core residue was not in a of side-chain of and and structures of the chains of of the subunit are and the protein surface and of the and of and whereas the hydrophobic core and with a in the of the of surface binding sites to that for a and receptors in which GI binding and are determined by to the receptors in which GI not binding in the for the affinity and the of sites with determined by to the The modeling the between and in and which and The α subunit in this which to A. R. A. M. R. A. and New Scholar). model the in the a second of was using sequence in this region and of of the was an and we structures that we in this and equivalent residues in other to the hydrophobic core to for the that subunits we structures in which residues in the from the hydrophobic included and we and to of determined in A. J. 1995; Full Text Full Text PDF PubMed Scopus Google and of or determined from of binding S. H. Sine S.M. 2000; PubMed Scopus Google Scholar). are in many and to the structure for GI and for lysine and receptors GI GI are of to the data and are and the of sites to which to the are of the data and are and the of sites with A. The of receptors as and to the in a GI are of to the data and are and the of sites to which to the are of the data and are and the of sites with A. The of receptors as and to the we two of using the S. Scopus Google Scholar), The first the of of The second the protein of side The structure was using and residues to lysine along a from of the subunit from adult human AChR. lysine was with and subunit in by of surface receptors using the ligand The results a of with as lysine advanced along the protein for residues that for whereas for residues that for results from subunits that and whereas results from of the to receptors K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. T. J. Full Text PDF PubMed Google K. M. M. N. J. Sine S. J. 1999; PubMed Scopus Google Scholar). These results provide that residues in the AChR subunit form a lysine a subunit that can with the the of receptor distinguish between of the subunit and we of the that ligand binding of the receptor subunit K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. T. J. Full Text PDF PubMed Google Scholar, J.P. Neuron. Full Text PDF PubMed Scopus Google Scholar). for each we binding of ACh and α-conotoxin GI by the of the subunit a for the whereas receptors the subunit a with a half over of in ACh K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. M. N. J. Sine S. J. 1999; PubMed Scopus Google results are with the receptors a with two whereas receptors a affinity by a over of of GI for ACh and GI distinguish receptors the subunit from with the the lysine ACh and GI binding of the ligand in the are the residues in this region to the ligand binding site N. Sine S. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). lysine to of receptors with the of and results that lysine of subunits whereas lysine to subunits to lysine in a in the to of the binding as for However, for the the to of the binding sites a of to a of and the subunit of the of binding sites of of of which the The together with the in the for a in the human subunit K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). the the of the subunit to are to in of AChBP, which the of that form a delimiting a hydrophobic core and the protein surface the other (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 of subunits with lysine to with subunits to a large for a side in a hydrophobic together with the that to it in therefore that residues in the subunit form a structures of the from model of the subunit and the in of the residues to lysine are in whereas to are in of AChBP, residues the hydrophobic core are whereas the protein surface are in results that residues to lysine mutagenesis their side chains the hydrophobic whereas to lysine mutagenesis their side chains the protein the results alignment of equivalent residues between the subunit and key residues to the ligand binding site in the and align with their in and lysine scanning and ligand measurements to the regions in the subunit that form we a to distinguish receptors that the subunit from that do not that are as binding sites in the for together with by GI the of that are as of together with the for GI of lysine the between that and that do not However, results and for GI to from that for receptors the in the from to of the of the as for the in of the a with affinity for the of receptors the subunit along with of this the GI data are well by the of of and a and that subunits in this are in surface we measurements using the which to the site with The measurements a affinity of the along with a affinity of the site and of and affinity determined from the the are to the from the GI measurements measurements using of with and The and These of and of receptors the subunit to with the subunit These of for a in the human subunit K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar), the as The and with the and receptors other and in to AChR yet for each of residues contributes to the ligand binding site (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), the to binding of GI to the binding sites. that subunits we ACh The and to that subunits surface and The and to of the from that for receptors and four ligand surface of surface whereas subunits with or to surface for lysine not to the The to the in and are using the or two binding sites in second and are the of sites with and the not to the The to the in and in a are using the or two binding sites in second and are the of sites with and the to results for we surface or core side for with side chains are to the protein and and are to the protein the other of the and not by to their for subunit Sine S.M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). These are to the hydrophobic core of the as for the in (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 in homology modeling alignment of the sequence to with that of the determination of side-chain in the together with side-chain in AChBP, alignment of in the and between the the alignment of subunit and provides a basis for and with that of for of the between alignment are between AChR subunits and The and therefore the of in four of to which the homology modeling to A. J. PubMed Scopus Google Scholar, A. R. A. M. R. A. and New Scholar). The between and and an to in and of side-chain are the overall sequence alignment in modeling in the to a homology model of the extracellular domain of the adult human AChR using by between subunits their subunits The structure from was for between and in and by The overall mutagenesis results are structural model of the using to and to subunits The of and subunits in of the whereas which two and of the align as of a along the of the a that in a to a hydrophobic core and the protein surface the the half of the the mutagenesis of with to the of the of or residues forms a to the of the and the of the the core and surface side as in the of the in residues their side chains the protein These surface residues to regions the from the hydrophobic side chains the protein These regions of and to regions in in which the from the core (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). lysine scanning not alignment of side chains between the subunit and regions the from the hydrophobic core. a from the we that the side of the protein to the equivalent residue in AChBP, which the hydrophobic core structural the surface of that residues of their side chains the protein surface of an in in the of with the of as the the subunit surface of side chains to of from the hydrophobic core together with of the between the AChR subunit and this in heteromeric AChR whereas in AChR that residues to lysine mutagenesis the protein whereas residues to mutagenesis the protein core side-chain we the subunit regions the hydrophobic and The region contributes to the subunit with the α subunit to of two ACh binding sites and and the of and residues to lysine mutagenesis the protein of side chains that the are and whereas are hydrophilic or and to to the ligand binding J. Full Text Full Text PDF PubMed Scopus Google Scholar), Y. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), S.M. N. R. Neuron. 1995; 15: Full Text PDF PubMed Scopus Google Scholar), S.M. S. A. PubMed Scopus Google Scholar), and S.M. J. Full Text Full Text PDF PubMed Scopus Google Scholar), the protein residues to lysine mutagenesis the hydrophobic core. These core residues are and and The region the of and the These form the of the subunit the which in to the transmembrane large of residues in this region to to of and from the hydrophobic core. a of the protein toward the of in the and side chains to the of the for the of hydrophilic side chains the protein of surface residues in this region are and whereas the residues are and The region contributes to the of the subunit and the subunit not in ligand binding and and that lysine mutagenesis their side chains the protein with to the protein surface yet as to lysine to of and and of to between the lysine side and the which the side of the surface side chains in the are or to the hydrophobic and and The surface side chains are hydrophobic and and residues to lysine mutagenesis their side chains the hydrophobic are and with and the hydrophilic model of the AChR ligand binding domain between with the of each subunit and that the of subunits regions of the and of the residues in each region the subunit with the to a of and the region of model a between and and between equivalent residues each subunit These are from the in (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), which forms between side chains from regions of the of a each AChR subunit the in of and AChR with as the the four in of and heteromeric AChR These stabilize the pentamer in or and are for and as for in proteins M. of and in Scholar). The region of to the ACh binding which are from the of the pentamer and These are and subunit as by site-directed and the The of the ACh binding and hydrophobic side chains from each The and the of the binding and are by the hydrophobic residues and and line the of the binding site of or their and and binding structures for ACh and form a and to the of the binding which not contributes to of for the over the it from in the N. Sine S. J. Full Text Full Text PDF PubMed Scopus Google Scholar). and equivalent residue in the AChR to of for the over the site S.M. S. A. PubMed Scopus Google Scholar). These and other residues the binding site structures that ACh binding to the of the in a The region of residue that form the the residues and whereas equivalent residues are the that forms the in AChR the protein surface from and in the N-terminal to and are to form an the from this the of the of to which it form a and are in of the AChR ligand binding interfaces. lysine scanning mutagenesis as a method to repeating structures that hydrophobic from hydrophilic regions in proteins. The method of of the side in a hydrophobic and of in the of the protein that the AChR subunit that a hydrophobic core and side-chain with in the homologous (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). results of side chains between in the subunit and in AChBP, regions in the and structural between and the AChR. experimentally determined sequence alignment provides the basis for a homology model using from the The resulting structural model provides a picture of the ACh binding site of the adult human which heteromeric and residues that from residues in equivalent in The model residue pairs subunit which as for by The structural model provides that can by mutagenesis to the structure and provide The overall suggest that lysine scanning to structural of other members of the AChR as well of other proteins not to x-ray crystallography or NMR. in protein by other or lysine not to such in the protein the lysine in a repeating structure such as a to it in a structural that scanning mutagenesis to in human to H. K. M. M. J. 2000; Full Text Full Text PDF PubMed Google and to to sites for S. S. Pharmacol. Google Scholar). However, the the first to apply lysine scanning to distinguish core hydrophobic from surface hydrophilic environments of residue side for subunit the or of the structural by the subunit with subunits to form surface which do not to which a ligand binding K. M. N. S. Sine S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. M. N. J. Sine S. J. 1999; PubMed Scopus Google Scholar). The two ligand binding sites in the pentamer the of ACh and GI with binding site in receptors affinity for not to the of the which identification of The and with α and do not surface for and from that the of binding sites was in the of the with and subunits and the subunits for α and of of surface and receptors are of to the of a and a that allow the to from the hydrophobic core. structural model of the AChR extracellular domain as well as with the 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). results side-chain alignment of the regions are the between the two proteins. However, side-chain in a of which we structural model are in the and are in non-α which or in AChBP, and in the α which in this region are in the ligand binding sites residues in heteromeric and from the residues in the AChBP, and and are in between the between and and between and the the are in the α which residue in the non-α which to the are in the α which a residue and the and in AChBP, and in non-α which to residues and The ACh binding sites are and subunit as by site-directed and the Mutagenesis and site-directed labeling that in the to the ACh binding and in the α subunit and and in or 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). The structure that residues in each of are the ACh binding and model provides a picture of and residues in the human AChR. the residues in each of the to the ligand binding in in and in in in and in and in of residues and structures for ACh by mutagenesis and are the of the binding These in S.M. K. A. M. Neuron. 1995; 15: Full Text PDF PubMed Scopus Google and in S. H. Sine S.M. 2000; PubMed Scopus Google Scholar, H. Sine S.M. R. J. Full Text PDF PubMed Google in S.M. N. R. Neuron. 1995; 15: Full Text PDF PubMed Scopus Google Scholar, N. Sine S.M. J. 2000; Full Text Full Text PDF PubMed Scopus Google in N. Sine S. J. Full Text Full Text PDF PubMed Scopus Google and in S.M. S. A. PubMed Scopus Google Scholar, S.M. N. R. Neuron. 1995; 15: Full Text PDF PubMed Scopus Google Scholar, Sine S.M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. 2000; Full Text Full Text PDF PubMed Scopus Google and in S. H. Sine S.M. 2000; PubMed Scopus Google Scholar, H. S. J. Sine S.M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These residues structures for binding ACh the or for ACh with to the their in the ACh binding subunit interfaces are the in subunit to N. Nature. 1995; PubMed Scopus Google Scholar). can or and or The are for and between functional whereas the provide between subunits in or the model a between and as well as between equivalent residues each subunit The in the AChR are from the in (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), which form between side chains from regions of the in the AChR provide in functional or and therefore as for and or to that and of proteins M. of and in Scholar). results provide for AChR structure with subunit structure from model of the that each subunit regions that are the as well as regions that are and structures the the region of in structures the ligand binding which are and interfaces. the subunit interfaces to the ACh binding residues are and to the to to a in α whereas in and a functional for a between model of the AChR extracellular domain to are the of in of the between and the and The between and using between ligand and residue side combined with measurements of ligand binding of The from not in the as the hydrophobic by lysine results from are not by structural the surface yet side to the protein that the to the residue the side of the which to scanning to other intractable proteins. allow side-chain to determined in other regions of the AChR and are to and allow of the lysine side the chains in and the side scanning for the AChR and subunits as well as subunits in other members of the AChR such as and the in structural of the many intractable proteins to from and an protein homologous to a protein with repeating structures delimiting a hydrophobic secondary structure and side can determined by lysine scanning with a of protein The results allow alignment of model and and of homology Future studies structural model of the AChR ligand binding These by structures are with the of their are with the structural model and by to provide of the a structural model of ligand with the ACh binding site the of in the AChR.