Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8111-8115, September 1991 Neurobiology Molecular dynamics of dopamine at the D2 receptor (guanine nucleotide-binding regulatory protein-coupled receptors/three-dimensional structure/electrostatic potentials! molecular modeling) SVEIN G. DAHL*, 0YVIND EDVARDSEN, AND INGEBRIGT SYLTE Department of Pharmacology, Institute of Medical Biology, University of Troms0, N-9001 Troms0, Norway Communicated by Jean-Marie Lehn, May 20, 1991 (received for review January 28, 1991) ABSTRACT A three-dimensional model of the dopamine D2 receptor, assumed to be a target of antipsychotic drug action, was constructed from its amino acid sequence. The model was based on structural similarities within the super- family of guanine nucleotide-binding regulatory (G) protein- coupled neuroreceptors and has seven a-helical transmem- brane segments that form a central core with a putative ligand-binding site. The space between two residues postulated to be involved in agonist binding, Asp-80 and Asn-390, per- fectly accommodated an anti-dopamine molecule. Molecular electrostatic potentials were mainly negative on the synaptic side of the receptor model and around aspartate residues lining the central core and positive in the cytoplasmic domains. The docking of dopamine into a postulated binding site was exam- ined by molecular dynamics simulation. The protonated amino group became oriented toward negatively charged aspartate residues in helix 2 and helix 3, whereas the dopamine molecule fluctuated rapidly between different anti and gauche confor- mations during the simulation. The receptor model suggests that protonated ligands are attracted to the binding site by electrostatic forces and that protonated agonists may induce conformational changes in the receptor, leading to G-protein activation, by increasing the electrostatic potentials near Asp- 80. Cloning and sequencing of neurotransmitter receptors has demonstrated that they may be divided into various "super- families." The dopamine D2 receptor belongs to the super- family of receptors transferring signals into cells through guanine nucleotide-binding regulatory (G) proteins. Antago- nism of postsynaptic D2 receptors has long been regarded as the primary mechanism of action of antipsychotic drugs, and it has recently been proposed that also dopamine D1 (1), D3 (2), and D4 (3) receptors and serotonin 5-HT2 receptors (4) may be involved in antipsychotic drug action. Knowledge of the three-dimensional structure of such receptors would significantly add to our understanding of their molecular mechanisms and be useful in the search for drugs that affect these receptors. Amino acid sequences of more than 10,000 proteins are known, but only some 450 three-dimensional protein struc- tures have been reported. No detailed three-dimensional crystal structure of a neurotransmitter receptor molecule is yet available, to our knowledge. In the absence of such data, we have constructed a model of the D2 receptor and used it to simulate the molecular dynamics of dopamine-receptor interactions. Based on structural similarities within the su- perfamily of G-protein-coupled neuroreceptors, the model was constructed from the rat D2 receptor sequence (5) and refined by molecular dynamics simulations and molecular mechanics energy minimization. The modeling was based on the following five hypotheses: (i) Membrane-spanning domains of G-protein-coupled neurotransmitter receptors are a-helices. Hydropathy indi- ces along the peptide chains show that G-protein-coupled receptors contain seven hydrophobic domains, usually as- sumed to correspond to seven transmembrane stretches of residues (6). Electron diffraction (7) and electron microscopy (8) experiments have demonstrated that bacteriorhodopsin has seven a-helical segments spanning the cell membrane. All available biological and chemical data indicate also that the membrane-spanning domains of visual rhodopsin form a-hel- ices (9, 10). G-protein-coupled neurotransmitter receptors share some amino acid homology with visual rhodopsin, particularly in the seven hydrophobic domains (11) that most likely are a-helices. (ii) Each transmembrane helix contains 27 amino acids. An a-helix with 27 amino acids has a total length of =40 A and may traverse the cell membrane in an approximately perpen- dicular orientation. The 11 membrane-spanning a-helices in the crystal structure of the photosynthetic reaction center of Rhodopseudomonas viridis contain on average 27 amino acids (12). (iii) Membrane-spanning segments of G-protein-coupled neurotransmitter receptors have similar localizations in aligned protein sequences. Hydropathy indices provide only approximate locations of transmembrane segments in the peptide chain of a protein, and for one and the same receptor such locations often differ by several amino acids among different reports. Since G-protein-coupled neurotransmitter receptors show 50-75% sequence homology in the putative transmembrane domains, we assumed that the segments of all receptors corresponding to each a-helix would be placed in matching positions by sequence alignment, as indicated in Fig. 1. Presumably, this enabled more precise prediction of the positions of the transmembrane helices in the peptide chains, based on average hydropathy indices of 14 neurore- ceptors from the same superfamily. (iv) The most polar surface areas of the transmembrane helices form a central core. The seven a-helical segments in bacteriorhodopsin are roughly perpendicular to the cell mem- brane, slightly inclined to one another at various angles up to 200, and closely packed to form an oval ring structure (7, 8). Site-directed mutagenesis experiments have suggested that ligands bind to the putative transmembrane regions of 132- adrenergic (12, 28, 29) and muscarinic ml receptors (30), and it has been postulated that f-adrenergic receptors form a rhodopsin-like core containing a ligand binding site (28). The similarities in their primary structures indicate that this also may be the case for other G-protein-coupled neuroreceptors. (v) G-protein-coupled neurotransmitter receptors have a common ligand-binding site. Site-directed mutagenesis ex- periments have suggested that agonists and antagonists in- teract with Asp-113 and that agonists but not antagonists Abbreviation: G protein, guanine nucleotide-binding regulatory pro- tein. *To whom reprint requests should be addressed. 8111 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on December 17, 2020
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Proc. Nati. Acad. Sci. USAVol. 88, pp. 8111-8115, September 1991Neurobiology
Molecular dynamics of dopamine at the D2 receptor(guanine nucleotide-binding regulatory protein-coupled receptors/three-dimensional structure/electrostatic potentials!molecular modeling)
SVEIN G. DAHL*, 0YVIND EDVARDSEN, AND INGEBRIGT SYLTEDepartment of Pharmacology, Institute of Medical Biology, University of Troms0, N-9001 Troms0, Norway
Communicated by Jean-Marie Lehn, May 20, 1991 (received for review January 28, 1991)
ABSTRACT A three-dimensional model of the dopamineD2 receptor, assumed to be a target of antipsychotic drugaction, was constructed from its amino acid sequence. Themodel was based on structural similarities within the super-family of guanine nucleotide-binding regulatory (G) protein-coupled neuroreceptors and has seven a-helical transmem-brane segments that form a central core with a putativeligand-binding site. The space between two residues postulatedto be involved in agonist binding, Asp-80 and Asn-390, per-fectly accommodated an anti-dopamine molecule. Molecularelectrostatic potentials were mainly negative on the synapticside of the receptor model and around aspartate residues liningthe central core and positive in the cytoplasmic domains. Thedocking of dopamine into a postulated binding site was exam-ined by molecular dynamics simulation. The protonated aminogroup became oriented toward negatively charged aspartateresidues in helix 2 and helix 3, whereas the dopamine moleculefluctuated rapidly between different anti and gauche confor-mations during the simulation. The receptor model suggeststhat protonated ligands are attracted to the binding site byelectrostatic forces and that protonated agonists may induceconformational changes in the receptor, leading to G-proteinactivation, by increasing the electrostatic potentials near Asp-80.
Cloning and sequencing of neurotransmitter receptors hasdemonstrated that they may be divided into various "super-families." The dopamine D2 receptor belongs to the super-family of receptors transferring signals into cells throughguanine nucleotide-binding regulatory (G) proteins. Antago-nism of postsynaptic D2 receptors has long been regarded asthe primary mechanism of action of antipsychotic drugs, andit has recently been proposed that also dopamine D1 (1), D3(2), and D4 (3) receptors and serotonin 5-HT2 receptors (4)may be involved in antipsychotic drug action. Knowledge ofthe three-dimensional structure of such receptors wouldsignificantly add to our understanding of their molecularmechanisms and be useful in the search for drugs that affectthese receptors.Amino acid sequences of more than 10,000 proteins are
known, but only some 450 three-dimensional protein struc-tures have been reported. No detailed three-dimensionalcrystal structure of a neurotransmitter receptor molecule isyet available, to our knowledge. In the absence of such data,we have constructed a model of the D2 receptor and used itto simulate the molecular dynamics of dopamine-receptorinteractions. Based on structural similarities within the su-perfamily of G-protein-coupled neuroreceptors, the modelwas constructed from the rat D2 receptor sequence (5) andrefined by molecular dynamics simulations and molecularmechanics energy minimization. The modeling was based onthe following five hypotheses:
(i) Membrane-spanning domains of G-protein-coupledneurotransmitter receptors are a-helices. Hydropathy indi-ces along the peptide chains show that G-protein-coupledreceptors contain seven hydrophobic domains, usually as-sumed to correspond to seven transmembrane stretches ofresidues (6). Electron diffraction (7) and electron microscopy(8) experiments have demonstrated that bacteriorhodopsinhas seven a-helical segments spanning the cell membrane. Allavailable biological and chemical data indicate also that themembrane-spanning domains of visual rhodopsin form a-hel-ices (9, 10). G-protein-coupled neurotransmitter receptorsshare some amino acid homology with visual rhodopsin,particularly in the seven hydrophobic domains (11) that mostlikely are a-helices.
(ii) Each transmembrane helix contains 27amino acids. Ana-helix with 27 amino acids has a total length of =40 A andmay traverse the cell membrane in an approximately perpen-dicular orientation. The 11 membrane-spanning a-helices inthe crystal structure of the photosynthetic reaction center ofRhodopseudomonas viridis contain on average 27 aminoacids (12).
(iii) Membrane-spanning segments of G-protein-coupledneurotransmitter receptors have similar localizations inaligned protein sequences. Hydropathy indices provide onlyapproximate locations of transmembrane segments in thepeptide chain of a protein, and for one and the same receptorsuch locations often differ by several amino acids amongdifferent reports. Since G-protein-coupled neurotransmitterreceptors show 50-75% sequence homology in the putativetransmembrane domains, we assumed that the segments of allreceptors corresponding to each a-helix would be placed inmatching positions by sequence alignment, as indicated inFig. 1. Presumably, this enabled more precise prediction ofthe positions of the transmembrane helices in the peptidechains, based on average hydropathy indices of 14 neurore-ceptors from the same superfamily.
(iv) The most polar surface areas of the transmembranehelicesform a central core. The seven a-helical segments inbacteriorhodopsin are roughly perpendicular to the cell mem-brane, slightly inclined to one another at various angles up to200, and closely packed to form an oval ring structure (7, 8).Site-directed mutagenesis experiments have suggested thatligands bind to the putative transmembrane regions of 132-adrenergic (12, 28, 29) and muscarinic ml receptors (30), andit has been postulated that f-adrenergic receptors form arhodopsin-like core containing a ligand binding site (28). Thesimilarities in their primary structures indicate that this alsomay be the case for other G-protein-coupled neuroreceptors.
(v) G-protein-coupled neurotransmitter receptors have acommon ligand-binding site. Site-directed mutagenesis ex-periments have suggested that agonists and antagonists in-teract with Asp-113 and that agonists but not antagonists
Abbreviation: G protein, guanine nucleotide-binding regulatory pro-tein.*To whom reprint requests should be addressed.
8111
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
KGQKPRGKEQLAKRKTFSLVK ... ........HKKAARTLSAI LLAFIVTWTPYNIMVLVSTFCKD. ....VPETLWELGYWLCYVNSTINPMCYALCNKAFRDTFRLLLLL R ..... . .'4SGQNGDEKQNIVARKIVKMTKQPAKKKPPPSRIEKKVTRTI LAILLAFI ITVAPYNVMVLINTFCAP . C ..... IPNTVWTIGYWLCYINSTINPACYALCNATFKKTFKHLLMCiYKNIOC;TPAGMRPAANVARKFASIARNOVRKKRQMAARERKVTRTIFAI LLAFI LTWTPYNVMVlVNTFCQS . C...TPDTVWSIGYWLCYVNSTTNPACYALCNATFKKTFNHII,LPQ.C )YRN'T
FIG. 1. Aligned amino acid sequences of G-protein-coupled neurotransmitter receptors. Dl, human dopamine D1 receptor (13-15); D2 andD3, rat dopamine D2 (5) and D3 (2) receptors, respectively; 5-HTla, human 5-HT1a receptor (16, 17); 5-HTlc, rat 5-HT1, receptor (18); 5-HT2,rat 5-HT2 receptor (19); alb, hamster alb-adrenergic receptor (20); a2a, human au-adrenergic receptor (21); a2b, human a2b-adrenergic receptor(22); P1, human fi-adrenergic receptor (23); ,2, human f32-adrenergic receptor (24); Ml, porcine muscarinic ml receptor (25); M2, porcinemuscarinic m2 receptor (26); M4, human muscarinic m4 receptor (27).
interact with Asp-79, Asp-130, and Asn-318 (29, 31) andSer-204 and Ser-207 (32) in the (82 receptor. The correspond-ing aspartic residues also seem to be involved in ligandbinding to muscarinic ml receptors (30). Asp-79, Asp-113,and Asp-130 in the ,2 receptor are conserved in all sequencesshown in Fig. 1 and in the dopamine D4 (4) and D5 (33)receptors, which suggests that these residues are involved inligand binding and signal transduction in all G-protein-coupled neuroreceptors.Asn-318 in the P2 receptor is conserved in the dopamine
receptors, muscarinic acetylcholine receptors, and adrener-gic receptors. Ser-204 and Ser-207 in helix 5 ofthe p2 receptorare conserved in the dopamine receptors. It is possible thatthe corresponding residues (Asn-390, Ser-194, and Ser-197)also are involved in agonist binding to D2 receptors.The D2 sequence, like all the other sequences shown in Fig.
1, contains prolines in transmembrane segments IV-VII.Proline often appears at the end of a-helices in proteins andis known as a "helix breaker." However, the M subunit ofthe photosynthetic reaction center of R. viridis has a prolinenear the middle of the third transmembrane helix (12), whichdemonstrates that a proline does not necessarily represent abreaking point of a transmembrane a-helix.
METHODSSequence AlignMent The sequence of the rat D2 receptor
(5) and the sequences of 13 other G-protein-coupled neuro-transmitter receptors were aligned by the Needleman-Wunsch method (34), with the Gap program ofthe Universityof Wisconsin Genetics Computer Group Sequence AnalysisSoftware Package (35). With a gap weight of 3.0, and a lengthweight of 0.1, hydropathy indices were calculated for eachsequence by two methods (36, 37), and average indices were
calculated from the aligned sequences. The 14 sequenceswere selected to get a balanced contribution from variousreceptor families to the average indices.
Receptor Modeling. Structural refinement by molecularmechanics energy minimization and molecular dynamicssimulations were done on a Cray X/MP-28 supercomputer,using the AMBER all-atom force field (38). The cut-off distancefor nonbonded interactions was 8 A. A distance-dependentdielectric function was used to mimic the dielectric dampingeffect of water on intramolecular interactions. Energy refine-ment was done by 500 cycles of steepest-descent minimiza-tion followed by 2000 cycles of full-conjugate gradient min-imization. Molecular dynamics simulations were performedat 310 K with a step length of 0.001 psec, after an initialequilibrium dynamics phase starting from 0.1 K. Molecularelectrostatic potentials 1.4 A outside the water-accessiblesurface were calculated with a distance-dependent dielectricfunction and a 10.0-A nonbonded cut-off radius.
Initial models of each transmembrane a-helix, includingthe side chains, were constructed with the MIDAS computergraphics programs (39) from the rat D2 sequence (5). Eachhelix was refined by energy minimization and its water-accessible surface (40) and molecular electrostatic potentials(39) were calculated. The helices were assembled on thecomputer graphics system such that the most polar region ofeach helix surface was oriented toward a central core. Thehelices were closely packed in an antiparallel arrangement,clockwise as viewed from outside the cell membrane, suchthat the water-accessible surface contained no holes betweenhelices after structure refinement.The seven-helix structure, without connecting loops, was
refined by energy minimization. The terminal segments andloops between helices were added, based on a method (41)that gives s50l correct predictions of secondary structures
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in proteins (42). Although other methods may give morecorrect secondary structure predictions (43-45), we did nothave any adequate method for modeling the 131-amino acidcytoplasmic loop between helix 5 and helix 6. Therefore, only7 amino acids of this loop, taken from the segments con-nected to helix 5 and helix 6, were included in the model andlinked to each other. Thus the segment of the third cytoplas-mic loop that differs between the two D2 receptor subtypes(5, 46-48) was not included in the model.The receptor model was refined by molecular mechanics
energy minimization. A dopamine molecule was then placedin the putative binding site, where it fit precisely into thespace between Asp-80 and Asn-390. The receptor-ligandcomplex was further refined by energy minimization, whichproduced only negligible changes in the receptor structure.Because the initial modeling of the extracellular and cyto-
plasmic domains had been based on fairly inaccurate sec-ondary structure predictions, these parts of the model werefurther refined by 2000 steps of molecular dynamics simula-tion at temperatures increasing from 0.1 K to 300 K, followedby energy minimization, while the transmembrane heliceswere kept fixed. This produced minor changes in the loopsbetween helices and more noticeable conformational changesin the N- and C-terminal segments. Finally, the water-accessible surface and molecular electrostatic potentialswere calculated for the whole receptor model.
RESULTSPrediction of Transmembrane Domains. Average hydrop-
athy indices of the 14 aligned receptor sequences are shownin Fig. 2. Plotting of areas under "windows" of 27 aminoacids under the average hydropathy index curves as a func-tion of amino acid number produced peaks that predicted thestarting point of each transmembrane helix. Hydropathyindices calculated by the method of Kyte and Doolittle (36)gave well-defined peaks for helices 1-6, whereas indicescalculated by the method ofHopp and Woods (37) defined thestarting points of helices 1, 4, 5, 6, and 7.Three-Dimensional Receptor Structure. The D2 receptor
model is shown in Fig. 3. After energy refinement, thetransmembrane a-helices remained approximately antiparal-lel, were slightly bent, and were somewhat closer together atthe cytoplasmic end than at the synaptic end. Two of theresidues postulated to be involved in agonist binding, Asp-80in helix 2 and Asn-390 in helix 7, were placed adjacent to each
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FIG. 2. Prediction of transmembrane helices in the D2 receptor.(A and B) Average of hydropathy indices of 14 neurotransmitterreceptors, calculated by the methods of Kyte and Doolittle (36) (A)and Hopp and Woods (37) (B), as a function of position in the alignedsequences. (C and D) Area under a 27-amino acid "window" in thehydropathy index curve to the left, as a function of its starting pointin the aligned sequences.
FIG. 3. Three-dimensional model of the rat dopamine D2 recep-tor, viewed in the plane ofthe cell membrane. (Left) a-Carbon atoms,red in the membrane-spanning domains and white at the synaptic(upper part) and intracellular (lower part) sides, and side chains ofAsp-80 in helix 2 (blue), Asp-114 in helix 3 (blue), and Asn-390 inhelix 7 (green). Asp-114 is closer to the synaptic membrane surfacethan Asp-80. (Right) The water-accessible molecular surface (dot-ted), color coded according to electrostatic potentials (e). Blue, e <-15 kcal/mol; white, -15 < e < 15 kcal/mol; red, e > 15 kcal/mol.
other at a distance that closely accommodated the energyminimized anti conformation of dopamine. Asp-114 in helix3 and Ser-194 and Ser-197 in helix 5 were placed nearer to thesynaptic membrane surface than Asp-80 and Asn-390. Pre-sumably, modeling of the other dopamine receptors by thesame procedure would place the corresponding residues insimilar geometrical positions.
Electrostatic Potentials. The molecular electrostatic poten-tials were based on the charges of all surrounding atomswithin the defined cut-off radius and depended on molecularconformation and on atomic charges. The potentials weremainly negative in the putative extracellular domains and inparts of the central core of the receptor model and positive inthe postulated cytoplasmic domains (Fig. 3). The lowestelectrostatic potentials in the central core were -40 kcal/mol(1 cal = 4.184 J) near Asp-80 in helix 2 and -37 kcal/mol nearAsp-114 in helix 3. Strongly positive electrostatic potentials,up to 38 kcal/mol, were also found in the area between helix4 and helix 5, extending from a level one helical turn belowAsp-80 and down into the second cytoplasmic loop.Molecular Conformations and Dynamics of Dopamine. In-
ternal movements in molecules occur on a fsec time scale.The molecular dynamics of protonated dopamine approach-ing the postulated receptor binding site was examined in an80-psec simulation where the receptor was kept in a fixedposition. The coordinates and energies were saved at 0.5-psec intervals. The simulation required 200 min of central-processing-unit time on the Cray computer.The electrostatic forces were not sufficient to attract the
dopamine molecule to the postulated binding site during thesimulation. Weak potentials (1.0 kcal), directing the neuro-
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transmitter toward the area between Asp-80 and Asn-390,therefore, were applied during the initial 65 psec of thesimulation. Constrained atomic distances were as follows:Dopamine nitrogen to Asp-80 carboxyl oxygen, 0-25 psec at25 A, 25-55 psec at 10 A, and 55-65 psec at 4.5 A; dopaminepara-oxygen to Asn-390 carbonyl oxygen, 40-55 psec at 10 Aand 55-65 psec at 4.5 A.The side chain of the dopamine molecule moved several
times from one side of the phenyl ring plane to the other sideand fluctuated rapidly between various anti and gaucheconformations throughout the simulation. Fig. 4 shows a partof the receptor model and some of the dopamine structuresthat were observed during the simulation. As the neurotrans-mitter moved down the central core, its positively chargedamino group became oriented toward negatively chargedaspartate residues (Asp-80 and Asp-114) in helices 2 and 3.The amino group of dopamine remained close to Asp-80during the final 15 psec ofthe simulation, after removal ofthedirecting force. Fig. 4 Lower shows a dopamine molecule ina postulated binding site between Asp-80 in helix 2 andAsn-390 in helix 7.
DISCUSSIONThe negative molecular electrostatic potentials around theN-terminal segment and the loops between helices 2 and 3,helices 4 and 5, and helices 6 and 7 support the hypothesis (49)that these represent synaptic domains of the receptor. Thelargely negative electrostatic potentials at the synaptic side
FIG. 4. Docking of dopamine into a postulated D2 receptorbinding site. (Upper) Selected structures from an 80-psec moleculardynamics simulation of dopamine approaching the binding site. Apart of helices 2, 3, 6, and 7 are shown with their connecting loops.(Lower) Energy-minimized anti conformation of dopamine betweenAsp-80 (blue) and Asn-390 (green). Water-accessible molecular sur-faces (dotted) are color coded according to electrostatic potentials asin Fig. 3.
indicate that protonated ligands are attracted to the receptorby electrostatic forces.
f32-Adrenergic receptors are coupled to stimulatory G (Gj)proteins in 11- to 15-residue segments located near thecytoplasmic surface of the cell membrane, at each end of thethird intracellular loop, and at the N-terminal part of thecytoplasmic tail (6). The cytoplasmic domains in the D2receptor model with strong positive electrostatic potentials(Fig. 3) closely corresponded to the parts of the /32 receptorthat are coupled to a G, protein. This suggests that positiveelectrostatic potentials around certain cytoplasmic domainsof the D2 receptor may be involved in G-protein interaction.Our calculations also support, by a completely differentapproach, the "positive-inside" rule for membrane proteins,which has been suggested from site-directed mutagenesisexperiments (50).
It is interesting to note that the electrostatic potentials werestrongly positive between helix 4 and helix 5, only 10 A fromAsp-80, which was surrounded by an equally strong negativeelectrostatic field. This indicates that agonist-inducedchanges in the electrostatic field may play a role in signaltransduction in the D2 receptor, as suggested for G-protein-coupled receptors in general (51). Such a process could beinitiated by binding of a protonated agonist to Asp-80 toneutralize its negative electrostatic field.Energy-minimized anti-dopamine fit perfectly into the
space between Asp-80 and Asn-390 (Fig. 4), in accordancewith the hypothesis that this is the active conformation (52).However, our molecular dynamics simulations indicate that,rather than obtaining a complete lock-and-key fit to the activesite while the neurotransmitter stays in this conformation, theinteraction starts with an electrostatic attraction between theprotonated amino group on the ligand and negatively chargedaspartate residues at the binding site, while the neurotrans-mitter moves between various conformations (Fig. 4). Sucha "zipper" mechanism whereby a flexible ligand binds to amacromolecule in several successive steps has been postu-lated from thermodynamic considerations (53). Still, theprevailing concept of ligand-receptor interactions has been alock-and-key mechanism requiring a specific conformation ofthe ligand. The present molecular dynamics simulation sup-ports the "zipper" type of mechanism for dopamine-receptor interactions.
Fig. 5 shows the antipsychotic drug cis-(Z)-chlorprothix-ene with its protonated dimethylamino group in close contactwith Asp-114 in helix 3. The localization of Asp-114 closer tothe synaptic membrane surface than Asp-80 and Asn-390suggests that neuroleptics act by preventing dopamine accessto its endogenous binding site and offers a simple stericexplanation of how Asp-80 and Asn-390 may interact with
FIG. 5. Molecular structure of cis-(Z)-chlorprothixene in a pos-tulated D2 receptor binding site, viewed from the synaptic side. Helix1 is at the bottom of the figure.
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agonists but not with antagonists. The dynamics of theligand-receptor system would still enable competitive bind-ing of agonists and antagonists.The calculations presented here were intended to present
an approximate overall model ofthe D2 receptor, which mightprovide further insight into its mechanisms. The model doesnot rule out other possible arrangements of the various partsof the receptor. It is possible that the seven helices may bemore tilted relative to each other, that the central core isslightly narrower, and that the seven helices are arranged inan anti-clockwise order viewed from outside the cell, assuggested for bacteriorhodopsin (8), and it is quite likely thatthe number of residues in each helix may differ from 27.
In any case, our calculations clearly indicate that, tounderstand the molecular mechanisms, neurotransmitter-receptor interactions should be regarded as dynamic pro-cesses and that electrostatic mechanisms may be importantfor ligand binding and signal transduction in the receptor. Thereceptor model may be used to design protein-engineeringexperiments to test its validity and provide further insightinto receptor mechanisms. It would be interesting, for in-stance, to examine the relevance of the positively chargedarea between helix 4 and helix 5 for transducer mechanisms,by site-directed mutagenesis experiments.The model also provides a tool to simulate the molecular
dynamics of ligand-receptor interactions. Although probablyinaccurate in many details, we feel that the receptor modelpresented here may provide some insight into the mecha-nisms of G-protein-coupled neurotransmitter receptors.
We thank Dr. T. Johansen for guidance in using the University ofWisconsin Genetics Computer Group programs. S.G.D. thanksUniversitd Rend Descartes and the Institut National de la Santd et dela Recherche Mddicale, France, for providing working facilitiesduring preparation of the manuscript. This work was supported bythe Norwegian Research Council for Science and the Humanities andthe Troms Fylkeskommune.
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