MolecularBioSystems

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Computational and Systems Biology themed issue

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Molecular modeling and docking studies of human 5-hydroxytryptamine

2A (5-HT2A) receptor for the identification of hotspots for ligand

bindingwzKaruppiah Kanagarajadurai,y Manoharan Malini,y Aditi Bhattacharya,Mitradas M. Panicker and Ramanathan Sowdhamini*

Received 31st March 2009, Accepted 12th August 2009

First published as an Advance Article on the web 8th September 2009

DOI: 10.1039/b906391a

The serotonergic system has been implicated in emotional and cognitive function. In particular,

5-HT2A (5-hydroxytrytamine receptor 2A) is attributed to a number of disorders like

schizophrenia, depression, eating disorders and anxiety. 5-HT2A, being a GPCR (G-protein

coupled receptor), is important in the pharmaceutical industry as a proven target for these

disorders. Despite their extensive clinical importance, the structural studies of this protein is

lacking due to difficulties in determining its crystal structure.

We have performed sequence analysis and molecular modeling of 5-HT2A that has revealed a set of

conserved residues and motifs considered to play an important role in maintaining structural

integrity and function of the receptor. The analysis also revealed a set of residues specific to the

receptor which distinguishes them from other members of the subclass and their orthologs. Further,

starting from the model structure of human 5-HT2A receptor, docking studies were attempted to

envisage how it might interact with eight of its ligands (such as serotonin, dopamine, DOI, LSD,

haloperidol, ketanserin, risperidone and clozapine). The binding studies of dopamine to 5-HT2A

receptor can bring up better understanding in the etiology of a number of neurological disorders

involving both these two receptors. Our sequence analysis and study of interactions of this receptor

with other ligands reveal additional residue hotspots such as Asn 363 and Tyr 370. The function of

these residues can be further analyzed by rational design of site-directed mutagenesis. Two distinct

binding sites are identified which could play important roles in ligand binding and signaling.

Introduction

G-protein coupled receptors (GPCRs) and their downstream

signaling partners constitute one of the largest class of molecular

targets contributing to any human disease. This fact is

emphasized in the central nervous system, wherein GCPRs that

bind neurotransmitters, apart from ion channels, are the key signal

transducers and are involved in almost every aspect of neural

function. Perhaps, the most extensive of all neurotransmitter-

mediated GPCR is the serotonergic system (serotonin or

5-hyroxytryptamine and its cognate receptors), which has been

implicated in the etiology of many mental disorders such as

depression, anxiety, schizophrenia, eating disorders, obsessive

compulsive disorder (OCD), migraine and panic disorder.

GPCRs have been exhaustively characterized in terms of

their pharmacology and downstream signaling cascades.

However, structural information about these integral

membrane proteins is still elusive. The GPCR transmembrane

systems in general represent a most challenging task for

structure determination, as their ‘greasy’ hydrophobic surfaces

do not readily make the regular intermolecular contacts

required for crystal formation. Despite these technical

challenges, a few GPCR crystal structures have been solved

after the initial bovine rhodopsin structure,1 two adrenergic

receptor structures,2–5 and most recently the adenosine 2A

structure.6 These have been crystallized as engineered or

bound receptors to limit their flexibility while retaining

functionality. Due to the extensive biomedical importance of

GPCRs, there have been attempts to model7–9 the three-

dimensional structure of these receptors, like angiotensin

receptors,10 vasopressin receptor,11 5-HT2C receptor,12 and

of their interactions with their ligands, for instance.

During the past 20 years or so, multiple 5-HT receptor

subtypes have been characterized ranging from 5-HT1 to

5-HT7.13 In particular, the 5-HT2 receptor family currently

comprises of three receptor subtypes, 5-HT2A, 5-HT2B and

5-HT2C receptors, which are similar in terms of their molecular

structure, pharmacology and signal transduction pathways.

The amino acid sequences of the 5-HT2 receptor family have a

high degree of homology within the seven transmembrane

domains but they are structurally distinct from other 5-HT

receptors.14 Of all the 5-HT receptor subtypes found in cortex,

National Centre for Biological Sciences (TIFR), GKVK Campus,Bellary Road, Bangalore-560065, India. E-mail: kanaks@ncbs.res.in,malinim@ncbs.res.in, aditi@ncbs.res.in, panic@ncbs.res.in,mini@ncbs.res.inw This article is part of a Molecular BioSystems themed issue onComputational and Systems Biology.z Electronic supplementary information (ESI) available: Multiplesequence alignments, Ramachandran plot, structural alignment onconserved residues and LIGPLOT representations. See DOI:10.1039/b906391ay Both have equally contributed to this work.

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1877–1888 | 1877

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

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the G-protein coupled 5-HT2A receptor has received extensive

attention in both physiological and pharmacological

experiments.15 This particular receptor has been found to bind

to a number of agonists, antagonists and inverse agonists

which in turn regulate the signaling via G-proteins. It has also

been shown that dopamine, considered a separate neuro-

transmitter with its own set of cognate GPCRs, can interact

with and activate the receptor initiating other effector path-

ways than the normal ones carried out by serotonin.16 In this

particular study, we have carried out docking of 5-HT2A

receptor with eight of its ligands, which are classified under

four categories, such as endogenous ligands (serotonin and

dopamine), synthetic agonists (DOI and LSD), antagonists

(haloperidol and ketanserin) and inverse agonists (clozapine

and risperidone). Two of the most used synthetic agonists to

the 5-HT2 receptor sub-family are DOI (4-iodo-2,5-dimethoxy-

phenylisopropylamine) and LSD (lysergic acid diethylamide),

a semi-synthetic psychedelic drug from the ergoline family.

Both are known to have hallucinogenic properties. In

addition, many commonly prescribed anti-psychotic drugs

serve as antagonists or inverse agonists to this receptor class.

The two antagonists used in this study include Haloperidol

4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-fluorophenyl)-

butan-1-one) an older typical antipsychotic used in the treatment

of schizophrenia and Ketanserin (3-{2-[4-(4-fluorobenzoyl)-

piperidin-1-yl]ethyl}quinazoline-2,4(1H,3H)-dione) an anti-

hypersensitive drug which binds with highest affinity to the

5-HT2A receptor and also used as a specific radioactive ligand

of the receptor in binding studies. Two other ligands which we

have used are the inverse agonists (also called as ‘atypical

antagonists’) of the receptor Clozapine 8-chloro-11-(4-methyl-

1-piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine which is atypical

antipsychotic, most effective in the treatment of schizophrenia

and Risperidone 4-[2-[4-(6-fluorobenzo[d]2,4isoxazol-3-yl)-1-

piperidyl]ethyl]-3-methyl-2,6-diazabicyclo[4.4.0]deca-1,3-

dien-5-one which is also an atypical antipsychotic drug used in

the treatment of schizophrenia and bipolar disorder in young

people.

Some functionally important residues, like D120 and D155,

were found to be important for the amine family of receptors

and are reported to be involved in binding of serotonin as well

as other ligands to its receptor 5-HT2A.17 Likewise, two

conserved tryptophans across the family (W336 and W367)

have been predicted to be involved in the binding of the

agonists of 5-HT2A.18–22 Mutation of conserved D155 was

reported to have lower affinity for serotonin, DOI, ketanserin,

mianserin, and spiperone, but not for LSD.17–21,23–25

It was initially proposed that at least one additional residue

in TM3 (S159) helps to anchor the charged terminal amine

moiety of serotonin and related ligands. Mutation of this Ser

(S159A) residue was reported to cause a 17.6-fold decrease

(1.2 kcal mol�1) in the receptor affinity to serotonin and

smaller changes in the affinities of N,N-9-dimethyl-5-HT

(bufotenine, 4-fold, 0.6 kcal mol�1) and LSD (1.4-fold,

0.15 kcal mol�1).25 It was also reported that the mutation of

Ser159 produces a 8–20 fold decrease in the binding of 5-HT,

5-MeOT, 4-HT.26 Detrimental effects on affinity, potency

and intrinsic activity were observed with Phe 339 mutant for

N-benzyl analogs, whereas N-substituted phenylalkylamines

and traditional agonists were only weakly affected and Phe

340 mutant had similar effects on almost all ligands.27 From

modelling studies, Ser 239 was found to act as a hydrogen

bond donor and Ser 242 acts as a hydrogen bond acceptor.

Ser 239 was predicted to engage oxygen substituents at either

4- or 5-posistion of tryptamine ligands and position-5 of

phenylalkyklamine ligands. Ser 242 seems to be engaged and

important for binding of polar ring substituted tryptamine

ligands.27 These serine residues, however, found to have little

effect in intrinsic activity.

Two independently derived molecular models have predicted

that a highly conserved tyrosine (Y370, TM7) has important,

but mechanistically distinct, roles in agonist binding.19 Tyr 370

has been proposed to have a direct role for DOM binding,23

and it was also suggested that Y370 actually stabilizes the

negative charge from Asp155.19 A mutation of Y370 (Y370A)

yielded a receptor with greatly diminished affinity for serotonin

and DOM (2,5-dimethoxy-4-methylamphetamine), but not for

a-methyl-5-HT or bufotenine.

Reports relating to intracellular signaling motifs present in

the 5-HT2A receptor are extensive and can indeed be classified

as an ‘interactome’.28 Relatively fewer attempts are reported

on modeling and docking of ligands with the extracellular

N-terminal domain. It may be surmised that given the wide

range of compounds that the receptor interacts with during

displacement studies, the ligand binding pocket might indeed

be dynamic and flexible. A number of mutagenesis studies

have been carried out, as discussed above, with respect to

binding site and the signaling of the 5-HT2A receptor.17–21,24,25

5-HT2A receptor is largely homologous and conserved across

several species. Still, a number of reports indicate that the

existence of small amino acid changes between isoforms are

responsible for significant changes in the function of the

receptor29 underlying the importance of detailed sequence

comparisons. In addition, modelling of human 5-HT2A receptor

and docking of known ligands were attempted due to its

important role implicated in a host of diseases, where future

drug design will be worthwhile. This article describes our

current examination of the sequence alignments and the

docked complexes of the 5-HT2A receptor in the process

of identification of structurally and functionally important

residues which would be further studied extensively by

mutagenesis in terms of ligand interaction, downstream

signaling and trafficking.

Results and discussion

Sequence analysis

Conservation of residues along the hierarchy of the GPCR

family with respect to 5-HT2A receptor was analyzed through

sequence comparison. The sequence alignments were performed

on human amine GPCR sequences (Fig. S1), human 5-HT

receptor sequences (Fig. S2) and 5-HT2A orthologous receptor

sequences (Fig. S3), which revealed a set of conserved residues

(Table 1) and motifs (Table 2). We have also compared the

human 5-HT2A sequence against the rat 5-HT2A sequence

(Fig. S4), and other rodent 5-HT2A sequences (Fig. S5).

1878 | Mol. BioSyst., 2009, 5, 1877–1888 This journal is �c The Royal Society of Chemistry 2009

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Similarly, human 5-HT2A sequence was compared with human

5-HT2B and human 5-HT2C sequences (Fig. S6).z

Human amine GPCR sequence alignment. The human amine

GPCR sequence alignment (42 sequences) includes sequences

from dopamine receptors, histamine receptors, adrenergic

receptors, muscarinic acetylcholine receptor, trace amine

associated receptors and 5-HT receptors. Sequences from 5-HT3

subtypes were not considered in this alignment, since they do

not belong to the GPCR class. The alignment was analyzed for

conservation of DRY and NPXXY motifs, and other con-

served residues. The analysis revealed the conservation of

three Tyr residues found at TM regions and the loop regions

at the cytoplasmic end of the receptor. The first important Tyr

residue, which is the part of the DRY motif of TM3, was

found to be conserved in all the sequences except the sequences

of TAAR2, TAAR3, DRD4 and HRH3 receptors (in which the

residue Tyr was replaced by Phe). The second Tyr residue was

found at the ICL2 region, a few residues away from the

DR[Y/F] motif and found to be conserved in all the human

amine GPCRs except in 5-HT2 receptor subtypes (where His

(H183 of 5-HT2A) replaced Tyr in 5-HT2A and 5-HT2B and

Ala replaced Tyr in 5-HT2C). The third Tyr (Y254 of 5-HT2A)

residue was observed to be conserved at the end of TM5 in all

amine GPCR sequences except HRH3 and HRH4 (where it is

replaced by Asn). The residues involved in the formation of

‘ionic lock’ in the intracellular region, implicated in one of the

major conformational changes between the inactive and active

form of bovine rhodopsin, were also found in the 5-HT2A

receptor sequence.30 Arg (R135 of bovine rhodopsin,

corresponds to R173 of 5-HT2A, part of DRY motif at TM3)

and Tyr (Y223 of bovine rhodopsin, corresponds toY254 of

Table 1 Amino acid conservation along the human 5-HT class of receptor sequences, human amine GPCR family of receptors and human GPCRsuperfamily

Secondary structureregion

Conserved residues from various set of alignments

5-HT receptor sequencealignment

Amine GPCR sequencealignment

Whole GPCR superfamily(literature reported)

TM1 N(92) Conserved ConservedTM2 N(110) — —

S(115) — —D(120) Conserved ConservedV(127) — —P(129) — —

ECL1 W(141) Conserved —TM3 C(148) Conserved —

D(155) Conserved —S(162) Conserved —I(163) — —I(169) — —D(172) Conserved —R(173) Conserved ConservedY(174) — —I(177) — —

TM4 W(200) Conserved ConservedTM5 F(243) Conserved —

P(246) Conserved ConservedY(254) — —

TM6 F(332) — —W(336) Conserved —P(338) Conserved ConservedF(339) — —F(340) — —

TM7 W(367) Conserved —G(369) — —Y(370) — —S(373) Conserved —N(376) Conserved —P(377) Conserved ConservedY(380) — —

Table 2 Conserved motifs obtained from various set of multiple sequence alignments

Structural region Reported GPCR motifs

Observed motifs in sequences

Human Amine GPCR Human 5-HT GPCR

TM2 — — V[M/L]PTM3 [D/E]RY DR[Y/F] TXSI, DRYTM5 — — F[Y/F]XPTM6 — WXP[F/Y] WXPFFTM7 [ND]PX2Y WX2[Y/W] SX2NPX2 [Y/H] WXGY, SX2NPX2Y

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1877–1888 | 1879

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5-HT2A residue at TM5.31,32 The motif, WXP[F/Y] at the sixth

TM helix was found to be conserved throughout the alignment

(please see Fig. S1z). A glutamate residue (E318 of 5-HT2A)

was found to be conserved at the beginning of the sixth TM

region in all human amine receptors, except in TAAR2,

TAAR3, HRH4 and 5-HT6R receptors (where residues Asp

and Ala replace this residue). Similarly a motif WXX[Y/W] at

the beginning of seventh TM helix was found to be conserved

throughout the alignment. Likewise an Asn residue positioned

just before SXXNPXX[Y/H] motif at TM7 was found to be

conserved in all human amine GPCR sequences except in

5-HT2 subclass receptors (where it was replaced by either

Ser in 5-HT2A and 5-HT2B or Cys in 5-HT2C) (Table 2).

The lowest and highest sequence identity between any two

sequences in this set was found to be between 19% and 80%,

respectively.

Human 5-HT receptor sequence alignment. 12 sequences

from human 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6 and

5-HT7 receptors and their subtypes were referred to as human

5-HT receptor sequences. The conserved residues and motifs

found from this alignment are reported in Table 1 and Table 2,

respectively. The lowest and highest sequence identity between

any two sequences in this set was between 22% and 63%,

respectively.

Alignment of 5-HT2A ortholog sequences. Orthologs of

5-HT2A receptor sequences (14 sequences) were aligned to

identify amino acid exchanges (Fig. S3z). The lowest and

highest sequence identity between any two sequences in this

set was between 70% and 99.7% respectively. The major

differences among the ortholog sequences were found at the

N- and C-terminal ends.

On the whole, the residues observed to be conserved from

these alignments were found to be localized within the TM

region of the receptors with an exception of Trp 141 found in

the first extracellular loop (Table 1).

Comparison of human 5-HT2A sequence against rat 5-HT2A.

A close comparison of sequences between the human and rat

(Rattus norvegieus) 5-HT2A receptor was performed (Fig. S4z),owing to the fact that the rat is an extensively studied model

organism. The sequence identity between the human and rat

5-HT2A receptor is 91%. Species-specific residues (Fig. 1a)

were found to be distributed more in the loop regions (except

three residues, Ala 82, Val 150 and Ser 242, that were mapped

on TM1, TM4 and TM5, respectively). The substitution of

Ser 242 in the human 5-HT2A by alanine in the rat sequence,

had previously been shown to increase the affinity of the

receptor towards ergolines and tryptamines suggesting that

such changes could be pharmacologically important.33

Comparison of human 5-HT2A sequence against rodents 5-HT2A.

The 5-HT2A sequences of rodents such as Rattus norvegieus,

Mus musculus, Cricetulus griesius, Chinese hamster and

Mesocricetus auratus were compared against the human

5-HT2A (Fig. S5z). The sequence identity of human 5-HT2A

with any rodent 5-HT2A compared here lies between 90%

and 92%. The species-specific residues identified from this

alignment are reported in the Fig. 1b.

Comparison of human 5-HT2A sequence against human

5-HT2B and 5-HT2C sequences. Detailed comparison of amino

acid exchanges were also performed across the subclasses,

5-HT2A, 5-HT2B and 5-HT2C (Fig. S6z). The sequence identityof human 5-HT2A with human 5-HT2B and 5-HT2C is 43%

and 54% respectively suggesting that the human 5-HT2A is

closer to 5-HT2C than 5-HT2B. Subclass-specific residues were

found to be distributed across the transmembrane and loop

regions (Fig. 1c) suggesting that such exchanges could be

crucial in maintaining the specific function. Interestingly, one

residue amongst these was Ser 242 which is also specific to

Human 5-HT2A when compared to the rat 5-HT2A sequence.

The other residues stated in Fig. 1c may also play important

roles in function there by distinguishing the human 5-HT2A

from the other two members of its subclass.

Molecular modeling and validation

The human b2 Adrenergic receptor (b2-AR) crystal structure

was used as a template to model the human 5-HT2A receptor.

The human 5-HT2A receptor shares 34% sequence identity

with human b2-AR. The sequence alignment was analysed to

check the preservation of conserved residues throughout the

alignment and motifs, such as DR[Y/F] at the C-terminal of

TM3 and NPXXY at TM7 (Fig. 2). The sequence alignment

was compared with the alignment used for modelling by other

groups.34–37 and our alignment showed very few differences

(detailed comparison is addressed in the ESIz). The lengths ofthe TM helices were equivalent with the recently published

model by Bruno and co-workers,35 except that the TM5 of our

model has eight residues longer at the cytoplasmic end.

Cys residues at TM3 and ECL2, thought to be involved in

the formation of a disulfide bond, were conserved between the

template and the query. The sequence identity of individual

TM helices and loop regions can be obtained from

Supplementary Table 1.z TM helices were found to be highly

conserved between the two receptor sequences, but the loop

regions were more variable.

The model structure after loop building and energy

minimization through SYBYL (Fig. 3) was validated using

PROCHECK. The PROCHECK results for the model,

excluding the loop regions, shows more than 98% of the

residues are in allowed regions (92.4% in the strictly allowed

region and 6.4% in partially allowed region) (Fig. S7az). Thefull-length structure (model with loops) shows more than 96%

of the residues in the allowed region (strictly allowed was

84.7% and partially allowed region was 11.7%) of the

Ramachandran plot (Fig. S7b). The final model also retains

the disulfide bond formed between the Cys residues of TM3

and ELC2. Results of VERIFY3D38 showed that more than

90% residues are in allowed regions (Fig. S8z). There are fewresidues falling under negative regions of VERIFY3D plot

and were found to be in N-terminal (residue range: 18–20 and

60–80), ICL2 (residue range: 177–185) and ELC2 (residue

range: 351–356) loop regions. The observed variations with

both the PROCHECK and VERIFY3D in the disallowed and

negative regions were mainly due to the loop regions where

there was low sequence identity between the template and

query; however, not much variation was seen in the ligand

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binding region.Moreover, the side chain orientations of conserved

residues (conserved at least in all class-A GPCRs) in TM helices of

our model, were found to be similar while superposing the model

with other available crystal structures (Fig. S10z).

Electrostatic charge distribution

Analysis of the electrostatic charge distribution revealed two

distinctly charged regions (Fig. 4). (a) A negatively charged

region was observed in the N-terminal region of the 5-HT2A as

observed in the b2-adrenergic structure.2 This negative charge

is predicted to facilitate the binding of the positively charged

amines. (b) A positively charged region towards the cyto-

plasmic end, ICL3 and C-terminal loop regions which was

also noticed in the template structure. But, we have also

noticed the presence of few negative charges at the cytoplasmic

regions, like glutamates at positions 455 and 466, in the final

model. There were no negatively charged regions observed in

the interface of TM3, TM4 and TM5, as reported in the

human b2-AR.

Fig. 1 Species-specific and subclass specific residues. (A) Species-specific residues of 5-HT2A to human and rat (Rattus norvegieus) were identified

from the pairwise alignment of their sequences (B) Specific residues of human 5-HT2A were compared against such sequences in rodents

(Rattus norvegieus, Mus musculus, Cricetulus griesius, Chinese hamster and Mesocricetus auratus). (C) Subclass specific residues between human

5-HT2A, 5-HT2B and 5-HT2C. The sequence alignment of all the three sets can be found in Fig. S4, S5 and S6, respectively.z

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Molecular docking

In order to identify residues potentially involved in ligand

binding and to analyze its functional importance, the receptor

was docked to various ligands which included two endogenous

ligands, two agonists, two antagonists and two inverse

agonists (atypical antipsychotics). The ligands were found to

dock with the receptor in various conformations and distinct

docking poses were realized after clustering of RMSD

structural deviation (2.0 A cutoff) each time from their initial

position. The number and binding energies of the generated

clusters (Fig. 5) varied with respect to the ligand. The extent of

energy differences of the various docking poses, measured as

standard deviation of the binding energy of the clusters were

not high (greater than 0.308 kcal mol�1). The highest standard

deviation was observed for haloperidol that also had the

highest number of clusters. Interestingly, DOI (the most

well-studied, synthetic, strong agonist for 5-HT2A) was found

to accommodate all the conformations into a single cluster.

Since the standard deviation of the binding energies of all the

ligands is within the standard deviation of the AutoDock force

field (2.5 kcal mol�1), the cluster which was more energetically

reasonable and encompassing a maximum number of

functionally important residues, already documented as

functional from the literature, was chosen for further analysis.

A number of residues reported to be involved in binding to

different ligands (Trp 151 in case of serotonin, Asp155 in case

of serotonin and ketanserin,39,40 Ser 159 in case of serotonin,25

Ser 207 in case of serotonin,41 Ser 239 in the case serotonin,

DOI and LSD,42 Phe 339 in case of serotonin and benzyl

analogs,42 Phe 340 in case of serotonin and other small classic

ligands,42,43 Trp 367 in case of all 5-HT2A ligands and Tyr 370

in case of serotonin, ketanserin and DOI) were observed close

to the docking poses (Fig. 6). The docking poses of these

ligands with their secondary structure is available in the

Fig. S11z). A few other residues were identified to interact

with the ligands from this study (Table 3).

Correlation of binding energy with experimentally determined

binding affinities. The binding energy obtained for each ligand

was found to correlate positively with the experimentally

determined binding affinities. The plots show a linear trend

with a correlation coefficient of �0.349 (Fig. S9) validating theapproach for docking of each of the ligands. Such correlation

coefficient values are accepted in the literature for training

docking algorithms and scoring schemes. Given the inherent

limitations in reproducing accurate docking poses from

computer algorithms and the heuristic approximations (such

as rigid ligand docking and the absence of lipids and solvents),

this correlation was merely to obtain confidence in our docking

protocol.

Endogenous ligands. In the case of endogenous ligands,

serotonin docked with slightly better binding energy

(�6.96 to �6.06) than dopamine (�6.66 to �5.76), as expected(data not shown). The synthetic agonists, antagonists and the

inverse agonists bind to the receptor with better binding

energy indicating better docking than the endogenous ligands.

The data therefore suggests them to be effective competitors

(Fig. 5).

The correlation of the residues in this study with the

literature-supported mutational and biochemical data also

validates the docking experiment. Serotonin binds to the

receptor forming hydrogen bonds with two residues Asp 155

and Tyr370 and is positioned within the transmembrane

region of TM3 and TM7. Tyr 370, which is already shown

to be conserved across the 5-HT class of receptors, is involved

Fig. 2 The final alignment used for modeling the 5-HT2A receptor.

The residues marked in green on each TM helix are conserved

throughout the GPCR family. The motifs (DRY and NPXXY),

conserved throughout the Class-A GPCR receptors, are covered by

green box. The motifs (TXSI, XFF, PFF and WXGY), covered by red

box, are conserved in all the 5-HT GPCR class except in 5-HT6

receptor. The dashed red line indicates the disulfide bridge formed

across the CYS residues in TM3 (C148) and ECL2 (C227).

Fig. 3 The energy minimized final structure of the 5-HT2A receptor.

TM helices are labeled for easy identification.

1882 | Mol. BioSyst., 2009, 5, 1877–1888 This journal is �c The Royal Society of Chemistry 2009

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in a direct hydrogen bonding interaction with serotonin in our

docking studies. This is consistent with the mutagenesis study

carried out by various groups.39,40 Dopamine, an endogenous

ligand, also bound to 5-HT2A, assumed a favorable docking

pose where the ligand is spatially proximate to residues in the

loop region (ECL2 and ECL3) and also residues from TM5,

TM6 and TM7. This differential positions of binding of the

two endogenous ligands supports the possibility that dopamine

could bind to 5-HT2A and activate different effector pathways,

as has been shown by Bhattacharyya and coworkers.16

Synthetic agonists. Synthetic agonists, DOI and LSD, were

found to be interacting with TM3, TM5, TM6, TM7, ECL2

and ECL3 (Fig. 6) with a few residues in common (which

include Ser239, Ser159, Asp 231, Phe339, Phe340, Val 235 and

Gly238) suggesting that these residues could be important for

the binding of these agonists to the receptor. In a mutagenesis

study,42 it has been shown that S239 is critical for DOI, where

it shown to have a shift of B3 fold reduction in the binding

affinity. Though there was no reduction in the binding affinity

reported for LSD with the S239A mutant, it had been shown

to have a shift of B3 fold reduction on potency.42 This study

suggest that the residue S239 may be important for secondary

intramolecular interactions that create an activated receptor

state, even when a ligand does not directly engage this residue.

Similarly, aromatic residue Phe340 was shown to have an

effect on DOI binding.43

Antagonists. The antagonists, ketanserin and haloperidol,

bind to TM3, TM5, TM6, TM7 and ECL2 with direct

interaction of residues Phe 339, Phe 340, Ser 159, Tyr370,

Leu229, and Asp 155. Phe 339 and Phe 340 are involved in a

hydrophobic interaction with the ligands and also found to be

conserved across the 5-HT class of receptors. The residue

Ser159 formed H-bond with the ligand ketanserin, which is

in consistent with the interaction study carried out by Runyon

and co-workers.44

Inverse agonists. The docking solutions for the inverse

agonists (clozapine and risperidone), on the other hand,

suggest involvement of residues from TM3, TM5, TM6,

ECL2 and ECL3 with direct interaction of few residues in

common such as Trp336, Phe399, Trpq151 and Tyr370.

These observations also suggest the position of ligand and a

preference of residues in certain helices depending on the type

of the ligand. The H-bond interactions for each of the ligands

involved were furnished in Table 3.

Fig. 4 Surface representation of 5-HT2A model electrostatics was represented by calculated charge from red (acidic residues; �10 kbT/ec) to blue

(basic residues; +10 kbT/ec) using a dielectric constant of 70. (A) Poisson–Boltzmann electrostatics was calculated using APBS plug-in in

PYMOL. As expected, the binding site entrance along with the N-terminal region is negatively charged to attract the ligand molecule, middle of the

TM region is positively charged and the TM region at the cytoplasmic part, ICL3 and C-terminal loop regions are highly positively charged.

The presence of negative charges at the bottom regions may be due to presence of Glutamates at positions 455 and 466. (B) Top view of the binding

site cleft involving TM2, TM3, TM4, TM5, TM6 and TM7 regions showing negative charges.

Fig. 5 Illustration of the binding energy of the chemically reasonable

docked conformation observed for each ligand. Ligand types are

marked in different colours: endogenous ligands in blue, agonists in

maroon, typical antagonists in olive green and inverse agonists

(atypical antagonists) in orange.

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Runyon and coworkers44 studied AMDA derivatives

for their interaction with 5-HT2A receptor and suggest two

distinct modes of interaction by the ligands. It is rewarding to

note, that in our docking study, the sites of interactions

of typical and atypical antagonists are in adjacent but not

coinciding sites. Further, these results also agree with recent

Fig. 6 Representation of docked poses for each ligand. Snapshots of interactions of 5ht2a with various ligands from autodock. Top panel:

endogenous ligands, serotonin (left) dopamine(right). Second: agonists DOI (left), LSD (right), Third: antagonists, ketanserin (left) haloperidol

(right), Bottom: inverse agonists, risperidone (left) clozapine (right).

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biophysical experiments on 5-HT2A homodimers.45 Recent

single site mutations of 5-HT4 receptors suggest that the active

form of the receptor might go through conformational transitions

or switches that do not seem to be required for the action of

agonists.46 Availability of interacting residues of different ligands

should provide avenues for the rational design of single mutation

experiments. Three-dimensional modeling and docking studies of

ligands should also enable us to study conformational changes at

the putative binding site using molecular dynamics and how this

might influence ligand binding.

Interestingly, our sequence analysis across the human 5-HT

GPCR sequences had revealed the presence of motifs WXPFF

(at TM6) andWXGY (at TM7) that involve Phe339, Phe340 and

Trp367 implicated in ligand binding. Furthermore, additional

motifs like TXSI (at TM3), F[Y/F]XP (at TM5) and NPXXY

(at TM7) are proximate to residues Ser159, Ser239 and Ser373,

that our docking studies show, are likely involved in ligand

binding (Table 3). Residue Ser373 is conserved throughout the

human amine GPCR class in comparison to Ser159, and Ser239

that were found to be class-specific. Such results provide avenues

to merge the sequence analysis results together with docking

studies in recognizing functionally important residues.

Sequence analysis using bioinformatics tools, homology-

related molecular modeling in combination with molecular

Table 3 Analysis of the docked conformations for hydrogen bonds, hydrophobic and polar interactions

Ligand

Interacting residuesa

Hydrogen bond residues Hydrophobic residues Hydrophilic Residues

Serotonin Asp155 Tyr370 Asp155

Tyr370 Trp151 —— Val366 —— Val156 —— Ile152 —

Dopamine Asp231 Leu362 Asp231Asp356 Ala346 Asp356— Val235 Asn343

DOI Ser159 Leu229 Ser159

Asp231 Val235 Asp231— Gly238 Ser239

— Phe339 Ser242

— Phe340 Asn343

LSD Ser239 Leu229 Asp231— Phe340 Asp155

— Val235 Ser159

— Trp151 Ser239

— Phe339 —— Val336 —— Val156 —— Gly238 —— Tyr370 —

Ketanserin Ser159 Leu229 Ser159

— Phe340 Asp155— Val235 Ser239

— Tyr370 Asp356— Phe339 —— Val366 —— Trp151 —

Haloperidol Asp155 Phe340 Asp155Tyr370 Leu229 Ser159

— Tyr370 —— Gly238 —— Val235 —— Val156 —— Phe339 —

Clozapine Tyr370 Val366 Ser131— C227 Asn363— Ile152 —— Leu229 —— Trp336 —— Phe339 —— Trp151 —— Tyr370 —

Risperidone Ser159 Phe339 Ser159

Leu229 Trp336 Asp155

— Phe340 Asp336— Trp151 Ser239

— Tyr370 —— Val336 —

a Residues already reported to be involved in binding are represented in bold.

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dynamics simulation methodologies can aid and often

complement the experimentally derived data. The structural

data from these approaches can not only be used for the

purpose of rationalizing structure-activity data on low-molecular

weight agonists and antagonists within the context of a

protein binding pocket, but also for a better understanding

of mutagenesis experiments, thus qualifying GPCR structural

models as useful to provide rationale for biochemical experiments.

Such approaches would serve as important and valid

communication platforms to establish functional links

between molecular biology, biophysics, bioinformatics and

organic chemistry in a highly efficient manner.

Methodology

Sequence analysis

Sequence alignments were performed on sets of sequences to

identify conserved residues and motifs, which could have

structural and or functional implications in the biological

systems. All the sequences used in the analysis were downloaded

from the SWISSPROT47 and NCBI (http://www.ncbi.nlm.nih.gov/)

database including the human 5-HT2A sequence (P28223). The

alignment of the entire human GPCR dataset which includes

1426 entries was obtained from the GPCRDB database48

(http://www.gpcr.org/7tm/). The sequence alignments of the

human amine GPCR sequences include 42 GPCR sequences

which belong to the amine family of receptors, the human

5-HT receptor sequences include the different subtypes of

the 5-HT class of receptors and 5-HT2A ortholog receptor

sequences includes 14 5-HT2A sequences from other organisms.

The 5-HT3 class of receptors was excluded from the alignment

of the 5-HT class as they are not characterized as GPCRs and

they belong to the ion channel family of receptors. We have

also compared human 5-HT2A receptor sequences against

sequences, of (ii) rat 5-HT2A. (ii) rodents 5-HT2A and (iii) human

5-HT2B and 5-HT2C, to identify species-specific and subclass-

specific residues. The alignments were constructed using

CLUSTALW49 (Version�1.83) and the alignments were

manually-edited using JALVIEW50 (Version 2.4) to retain

high equivalence of conserved regions. These alignments were

analyzed for the identification of amino acid conservation and

for the identification of class-specific residues and motifs.

Throughout the text, the residues are numbered according to

human 5-HT2A receptor sequence.

Molecular modeling and validation

The crystal structure of the human b2 adrenergic receptor2

(PDB ID: 2rh1) was used as a template for the construction of

the model since it was found to be a more suitable template in

comparison to bovine rhodopsin51 (PDB ID: 1u19) in terms of

sequence. Like 5-HT2A, the b2-AR is also an amine GPCR,

which is also more closely related in terms of sequence identity

than the bovine rhodopsin and other available crystal

structures. The human 5-HT2A (ID: P28223) and the human b2adrenergic receptor (P07550) sequences was aligned using

CLUSTALW49 (Version�1.83) and edited using JALVIEW50

(Version 2.4). The TM regions were predicted for the human

5-HT2A receptor sequence using transmembrane prediction

servers, such as TMHMM,52 SOSUI,53 and HMMTOP,54 to

guide and improve the alignment in the TM regions.

The structure of the template was obtained from RSCB55

(http://www.rcsb.org/pdb). The coordinates corresponding to

1–28, 231–262, 343–365 segments were not available in the

template crystal structure due to poor electron density and

hence these residues were removed from the query sequence

before the alignment. The final alignment was used to

construct the model using the software MODELLER56

(Version 9.1). A set of 100 structures were generated, from

which the low energy structure was used for further processes.

The initial low energy model obtained from MODELLER

was validated by using PROCHECK57 server. The structure

was further energy minimized using the SYBYL software

package (Version 7.1) (Tripos Associates Inc.). Tripos force

field, using 500 iterations of steepest descent and 100 iterations

of conjugate gradient, with a distance dependent dielectric

constant equal to 1 and a non-bonded interaction cutoff

value of 8 and was terminated at a convergence of

0.05 kcal mol A�1. The energy minimized structure was further

utilized to build the N-terminal (1–70 residues), C-terminal

(396–471 residues) and the ICL3 (265–316 residues) regions

using pre-installed PRODAT database tool in SYBYL and

the structure was again energy minimized using the same

parameters successively after the construction of every loop

region. The final structure was validated again using the

PROCHECK57 and VERIFY3D38 server. We have also

analyzed the side chain orientation of conserved residues

(which are conserved at least in all class-A GPCR sequences),

by superposing our model against the available crystal

structures (bovine rhodopsin, turkey b1-AR, human b2-AR

and human A2A receptor) to analyze how good our model was

coinciding with other structures.

Electrostatic charge distribution

The Adaptive Poisson-Boltzmann Solver (APBS)61 program

was employed to calculate the electrostatic charge distribution

of 5-HT2A model structure. The consideration of a membrane

environment has been indirectly included by reducing the

dielectric constant to 70, as used in the b2 adrenergic structure2

to calculate the charges. The electrostatics were mapped on

the model using the molecular surface representation and was

compared with the electrostatic charge distribution of the

crystal structure of human b2-AR.

Molecular docking

Docking was performed using the program Autodock4.058

with the Lamarkian genetic algorithm to dock eight different

ligands which included two endogenous ligands (serotonin and

dopamine), two agonists (DOI59 and LSD25), two antagonists

(ketanserin60 and haloperidol62) and two inverse agonists

(clozapine62 and risperidone63). The binding cavity was

defined to cover the entire transmembrane region as a grid

of size 78*70*70 and a spacing of 0.375. The docking was

carried out, with flexible ligand, for 100 GA runs with a

population size of 150 with 25*105 evaluations and a maximum

of 27 000 generations. The conformations were analyzed based

on the generated clusters using the MGL Autodock tools

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(Version 1.5.2). The 3D structures of the ligands were

generated using the molecular design suite (MDS; VLife

Sciences Pvt. Technologies Ltd.) with their initial coordinates

from the PUBCHEM database. The correlation of binding

energy of the ligands with their experimental binding affinities,

obtained from the literature (as mentioned above), was studied

using scatter plot.

Acronyms

5-HT2A 5-hydroxytrytamine receptor 2A

GPCRs G-protein coupled receptors

DOI 4-iodo-2,5-dimethoxyphenylisopropylamine

DOM 2,5-Dimethoxy-4-methylamphetamine

ECL Extracellular loop

ICL Intracellular loop

LSD Lysergic acid diethylamide

TM Transmembrane region

Acknowledgements

R.S. was a Senior Research Fellow of Wellcome Trust, U.K.

K.K. is supported by University Grant Commission, India.

A.B. and M.M.P. would like to thank Department of

Biotechnology, India for financial support. M.M. is supported

by Career Enhancement Award of R.S. by Department of

Biotechnology, India. Both M.M.P. and R.S. thank NCBS

(TIFR) for financial and infrastructural support.

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