Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor

166

Molecular Brain Research. 14 (1992) 166-178 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00

BRESM 70441

Molecular dynamics of serotonin and ritanserin interacting with the 5-HT 2 receptor Oyvind Edvardsen, Ingebrigt Sylte and Svein G. Dahl Department of Pharmacology, Institute of Medical Biology, University of Troms~, TromsO (Norway) (Accepted 18 February 1992)

Key words: Serotonin: Ritanseriu: Serotonin 5-HT2 receptor: Three-dimensional structure; Molecular modeling: Molecular dynamics: Electrostatic potential

A three-dimensional model of the serotonin (5-hydroxytrytamine" 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 (z helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor -- ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp t20. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations. INTRODUCTION The serotonin (5-hydroxytrytamine; 5-HT) 5-HT 2 receptor belongs to the superfamily of G protein-coupled neurotransmitter receptors, and utilizes phosphatidylinositol turnover as effector system 32. Radioligand binding experiments have indicated that the 5-HT 2 receptor both has a high-affinity state and a low-affinity state 2. It has previously been postulated that fl-adrenergic receptors ~ and G protein-coupled 5-HT receptors 19 have seven m e m b r a n e - s p a n n i n g a helices which form a rhodopsin-like core containing a ligand binding site. Sitedirected mutagenesis experiments with the fi2-adrenergic receptor have shown that antagonists bind to Asp 1133s''~ while agonists interact with Asp 79, Asp 113, Asn 31835` 3~,, Asp 130 u', Ser 204 and Ser 20737, all located in the putative t r a n s m e m b r a n e regions of the receptor. Corresponding aspartic residues in the muscarinic M~ receptor 17 (Asp 71 in t r a n s m e m b r a n e helix 2 and Asp 105 in t r a n s m e m b r a n e helix 3) and in the dopamine D2 receptor 3~ (Asp 80 in t r a n s m e m b r a n e helix 2) have also been suggested to be involved in agonist and antagonist binding. Asp 79 in helix 2, Asp 113 and Asp 130 in helix 3 of the f12 receptor are conserved in all known G proteincoupled neurotransmitter receptor sequences, which sug-

gests that the corresponding aspartic residues may be involved in ligand binding to all these receptors. Asp 79, Asp 113 and Asp 130 in the fi2 receptor corresponds to Asp 120, Asp 155 and Asp 172, respectively, in the 5-HT 2 receptor 7. We have previously constructed a model of the dopamine D x receptor 7. based on its amino acid sequence and on the structural similarities within the superfamily of G protein-coupled neurotransmitter receptors. We report here a three-dimensional model of the rat brain 5-HT~ receptor, constructed from its amino acid sequence :4 by similar techniques. Ritanserin is a potent and relatively selective 5-HT~ receptor antagonist 2v'>, structurally related to ketanserin. The interactions of 5-HT and ritanserin with the 5-HT 2 receptor model were examined by molecular dynamics simulations and molecular mechanics energy minimization. As in our previous study 7, the modeling of the 5-HT 2 receptor was based on several hypotheses: (i) The m e m b r a n e - s p a n n i n g parts of G protein-coupled neurotransmitter receptors form ct helices, assembled such that the most polar surface areas form a central core. (ii) Different G protein-coupled neurotransmitter re-

Correspondence: S.G. Dahl. Institute of Medical Biology, University of Troms~a, N-9001 Troms¢~, Norway.

167

TABLE I

Parameters used in A M B E R molecular mechanics energy minimization (EMIN) and molecular dynamics simulation (MD) *r = interatomic distance.

Cut-off distance for non-bonded interactions Scaling factor, 1-4 van der Waals interactions Scaling factor, electrostatic interactions Initial step length Step length Steps between updating of non-bonded pair list Dielectric function

EM1N

MD

12.0 ~ 2.0 2.0 0.05

12.0 /k 2.0 2.0

100

i0

1.0' 10 15 s ,£

c e p t o r s h a v e a c o m m o n l i g a n d b i n d i n g site in t h e c e n t r a l core. (iii) E a c h t r a n s m e m b r a n e helix has 27 a m i n o acid residues. (iv) M e m b r a n e - s p a n n i n g s e g m e n t s o f d i f f e r e n t G p r o t e i n - c o u p l e d n e u r o t r a n s m i t t e r r e c e p t o r s h a v e s i m i l a r loc a l i z a t i o n s in t h e a l i g n e d p r o t e i n s e q u e n c e s . B a c t e r i o r h o d o p s i n has s e v e n a - h e l i c a l s e g m e n t s s p a n n i n g t h e cell m e m b r a n e 1'21'22, a n d biological a n d c h e m ical d a t a i n d i c a t e t h a t also visual r h o d o p s i n has such an a r r a n g e m e n t o f s e v e n t r a n s m e m b r a n e s e g m e n t s tS. T h e r e is r e l a t i v e l y high h o m o l o g y b e t w e e n visual r h o d o p s i n and G protein-coupled n e u r o t r a n s m i t t e r receptors, particularly in t h e p r e s u m e d t r a n s m e m b r a n e p a r t s , w h i c h m o s t likely are ct h e l i c e s 29.

=

r*

C

=

r*

MATERIALS AND METHODS

Model building Initial model building was performed with the MIDAS molecular graphics computer programs 14 on an Evans & Sutherland PS390 computer with a MicroVAX II/Ultrix computer as the host system. The localizations of the seven transmembrane ct helices in the amino acid sequence of the 5-HT 2 receptor24 were determined from average hydropathy indices of 14 different G protein-coupled neuroreceptors, calculated after alignment of their amino acid sequences7. Initial models of each a helix were constructed with the Addaa module of the MIDAS programs. This procedure arranged all residues within the specified sequence in an ct helical structure with all phi, psi angles at -57 °, -47 °. Each a helix was refined by molecular mechanical energy minimization, using the AMBER 3.0 programs36. Water-accessible molecular surfaces6 and electrostatic potentials were then calculated for each helix. The seven ct helices were initially assembled in an anti-parallel, almost circular arrangement. The helices were ordered in an anticlockwise arrangement viewed from the N-terminal side, as suggested in a model of bacteriorhodopsin22. The surface of each he-

TABLE II

United atom force field parameters for ritanserin k r = bond stretching force constant, req = equilibrium bond length, k o = bending force constant. 0eq = equilibrium bond angle. V = rotational barrier. R = van der Waals radius, e = van der Waals potential well depth. The atom types are indicated in Fig. 1. X denotes any atom type.

Bond

k,. (kcal. m o F 1.~ - : )

re, (~ )

Torsion angle"

V/2 ( kcal. tool -I

$5-C5 C5-C5 CE-N5 N5-C5 S5-CE N5-C C -C6 C6-C6 C6-N6 N6-CE C6-C3 C6-C2 CZ-CZ CA-F C2-CZ CZ-CA

200 500 422 406 200 379 434 555 432 550 317 317 570 41712 317 317

1.738 1.335 1.385 1.397 1.728 1.417 1.428 1.346 1.377 1.288 1.510 1.510 1.340 1.360 1.510 1.510

X -CZ-CZ-X X -CZ-CA-X X -C2-CZ-X X -C5-N5-X X -CE-S5-X X -C6-C6-X X -N6-CE-X X -N5-C -X X -N6-C6-X X -C -C6-X X -N5-CE-X X -C2-C6-X C5-N5-CE-S5 $5-C5-C5-N5 CE-$5-C5-C5

30.0 0.0 0.0 4.6 30.0 5.2 28.4 0.9 5.4 5.6 5.2 0.0 5.5 30.0 30.0

168 lix was color coded according to the electrostatic potentials, which were used to orient each helix such that the most polar region was directed towards the central core of the receptor model. Models of the loops between helices and the C- and N-terminals were constructed with the Addaa program of the M I D A S package, with conformations based on secondary structure predictions by the Chou and Fasman method 45. Using the computer graphics system and the M I D A S programs, the loops and terminals were positioned near the ends of the t r a n s m e m b r a n e a helices by interactive graphics, and connected to the helices. A m i n o acid residues predicted to be in a random coil conformation were used to bend the loops such that they were positioned near the presumed planes of the synaptic and cytoplasmic m e m b r a n e surfaces, as observed in the photosynthetic reaction center of Rhodopseudomonas viridis ~~). The third cytoplasmic loop between helix 5 and helix 6 was truncated and contained only 10 amino acids from each end. These fragments were joined to form a loop of 20 amino acid residues, instead of the total length of 64 residues. ,]'ll'll('lltFe FefiHel;,zellt

Molecular mechanical energy calculations and molecular dynamics simulations were performed with the united atom force field of

T A B L E II (continued) Angle

ko (kcal.rnol-l.rad 2)

Oeq (°)

C5-S5-CE C5-N5-CE S5-CE-N5 N5-C5-C5 C5-C5-$5 CE-N5-C C5-N5-C N5-C -O C6-C - 0 C -C6-C6 C -C6-C2 C6-C6-N6 C6-N6-CE N6-CE-S5 N6-CE-N5 N5-C -C6 C6-C6-C3 C3-C6-N6 C6-C6-C2 C6-C2-C2 C2-N3-C2 C2-C2-CZ ('2-CZ-CZ C2-CZ-C2 CZ-CZ-CA CZ-CA-CD CA-CZ-CA F -CA-CD

55 90 60 70 90 85 85 80 811 85 85 7(1 711 70 70 70 85 85 70 63 511 63 70 711 85 85 85 713

90.9 114.4 110.1 112.2 111.9 123.2 122.4 123.4 125.3 121.5 118.5 123.8 114.4 121.8 126.0 I11.3 120.11 116.2 120.0 114.0 11/9.5 114.t) 120.11 120.0 120.0 120.0 120.11 121t.0

Non-bonded ~

R (Ji)

C5 N5 S5 C6 N6 CZ F

1,85 1,75 2,00 1.85 1.75 1.85 1,74 ts

" Phase shift angle = 180°. Periodicity = 2. b Van der Waals 1-4 interaction.

0.1211 0. 1611 0.20(/ 0. 120 0.1611 0. 1211 I). 154 Is

the A M B E R 3.0 computer programs ~4"41"4~-, with . the nitrogen atom in histidine partially protonated. The calculations were done on a Sun 4/60 workstation and on a Cray X-MP/216 supercomputer, using the parameters given in Table I. Water-accessible surfaces 6 and molecular electrostatic potentials 1.4 ,~ outside the surface were calculated with the M I D A S programs ~4, using a distance dependent dielectric function (e = r) and a I2.0 A non-bonded cut-off distancc. Energy minimization of 5-HT, ritanserin, and of individual cx helices was performed by the steepest descent method for the initial 10 steps and for the first 10 steps after updating of the non-bonded pair list, followed by conjugate gradient minimization until convergence with a 0.02 kcal mol ~/k ~ gradient difference between successive steps. The whole receptor model was refined by 500 cycles of steepest descent minimization, followed by 2000 steps of conjugate gradient minimization. The start model of the receptor, obtained by computer graphics methods, was first refined by energy minimization. The loops and terminals were then allowed to move during a 20 ps molecular dynamics simulation, while the helices were kept in fixed positions. The final coordinate set from this simulation was energy minimized in order to obtain an initial receptor model. Water-accessible surfaces and electrostatic potentials were then calculated for the receptor model. From the all atom representation of 5-HT used in a previous study t3, a united atom model of the lowest-energy conformer was constructed by incorporating all C-bonded hydrogen atoms into the carbon atom. Hydrogen atoms bonded to oxygen and nitrogen were represented explicitly. The atomic parameters of tryptophan 4~ were used for 5-HT, with two exceptions: The OH-substituted carbon atom in 5-HT was assigned atomic parameters as for tyrosine 4~, and standard A M B E R parameters of an aliphatic chain 4~ were used for the angle between two side chain carbon atoms and the ring carbon atom connected to the side chain. The A M B E R force field parameters for ritanserin are given in Table II*. Normal mode calculations and the crystal structures of /4 different 5H-thiazolo[3,2-a]pyrimidines and related compounds, obtained from the Cambridge Crystal Structure Database, were used to generate parameters for the fused ring system in ritanserin (Fig. I). For carbon-carbon and carbon-nitrogen bonds, equilibrium bond lengths were determined from the X-ray structures, and force constants and torsional parameters were calculated by interpolation as described by Weiner et al. 4t. Other bond force constants and all angle force constants for thiazole were determined by normal mode calculations. The remaining equilibrium bond angles were determined from X-ray structures of 5H-thiazolo[3,2a]pyrimidines, and bond force constants were determined from standard A M B E R parameters of structural elements similar to those in ritanserin. The energy minimized geometry of the 4-azacyclohexylidene fragment corresponded well to the crystal structures of substituted 4-azacyclohexylidenes e~. An all atom model of ritanserin, with a protonated piperidine nitrogen atom, was generated and energy minimized without including electrostatic interactions. A b initio electrostatic potential based atomic point charges were calculated with the Q U E S T 1.0 ~ ~4 program .... , using an STO-3G basis set. Atomic charges were calculated for two overlapping fragments of the ritanserin molecule, with the central - C H . - C H z- bridge present in each fragment. The charges were used in energy minimization of the ritanserin structure in vacuo, including electrostatic interactions, with the central -CH:-CH~- torsional angle in +gauche, -g'auche and anti orientations. Each energy minimized conformer of ritanserin was used as the start structure for a 105 ps molecular dynamics simulation in l'(ICltO.

* A compplete list of parameters and atomic charges used for 5-HT and ritanserin is availabl:e from the authors.

169

Molecular dynamics Classical molecular dynamics simulations without pressure monitoring were performed at 310 K. The initial temperature was 0.1 K, and the molecular system was assumed to be equilibrated within the first 5 ps of each simulation. All bond lengths were constrained during the simulations. The coordinates of the molecular model were saved at 0.5 ps intervals, and the torsional angles of the central -CH2-CH2-moiety of the ligands were calculated for each of the saved coordinate sets. The reported simulation times include the initial equilibration period. As in our previous simulations with an all atom model of 5-HT 13, swift conformational changes took place during molecular dynamics simulations of 5-HT in vacuo, using the united atom parameters and keeping all bond lengths constrained. However, the anti conformation of 5-HT was fairly stable during the united atom simulations with all bond lengths constrained, while only gauche conformers were observed during previous simulations with an all atom model of 5-HT and only bond lengths involving an hydrogen atom constrained 13. All molecular dynamics simulations of ligand-receptor interactions started from the initial, energy minimized receptor model. The ligand was placed in the start position with the computer graphics system, and the ligand-receptor system was energy minimized as described above, before the molecular dynamics simulation was started. After each simulation the receptor-ligand system was energy minimized and interaction energies of the receptorligand complex were calculated. During a 20 ps molecular dynamics simulation (A), the receptor model was kept in a fixed position while the 5-HT molecule was allowed free movement. The simulation was started from the lowest-energy (gauche) conformer of 5-HT 13, placed at the synaptic side of the receptor model. The lowest-energy conformer of 5-HT was then placed in the transmembrane duct with the amino group near Asp 120 in helix 2, and the hydroxyl group directed towards Asn 376 in helix 7. In a following 100 ps molecular dynamics simulation (B), the helices, loops between helices and the 5-HT molecule were allowed free movement, while the N- and C-terminals were kept in fixed positions, except for the three amino acids closest to helix 1 and helix 7. A protonated ritanserin molecule with the central -CH2-CH 2fragment in the lowest-energy (-gauche) conformation was placed

in the transmembrane duct of the initial energy refined receptor model. The piperidine nitrogen atom was placed near Asp 155 in helix 3, the fused ring system was directed towards helix 6, and the fluorobenzene groups were oriented towards the cytoplasmic end of the transmembrane duct. The molecular dynamics of the ritanserin-receptor complex was examined in a 100 ps simulation (C), where both the ligand and the receptor were allowed to move as in simulation B with 5-HT. The ritanserin molecule was again placed in the transmembrane duct of the initial receptor model, this time oriented such that the fiuorobenzene groups were directed towards the synaptic, end of the central duct and the fused ring system towards helix 4 and helix 5. The molecular dynamics of the ritanserin-receptor complex was examined in a 100 ps simulation (D), where the ligand and receptor were allowed to move as in simulations B and C. The final coordinate set from each simulation was energy minimized, the water-accessible surfaces and electrostatic potentials of the ligand-receptor complex were calculated, and the ligand-rcceptor interaction energies were calculated with a 12 A. cut-off radius. A 100 ps simulation of the ligand-receptor complex consumed approximately 5 weeks of CPU time on the Sun 4/60 computer and 40 h of CPU time on the Cray computer.

RESULTS

Charge distribution T h e a m i n o acids w h i c h m a y b e i o n i z e d at p h y s i o l o g i cal p H ( A s p , G l u , A r g , Lys a n d H i s ) w e r e m a i n l y loc a t e d in t h e s y n a p t i c a n d c y t o p l a s m i c d o m a i n s o f t h e rec e p t o r m o d e l , as s h o w n in Fig. 2. T h e s y n a p t i c d o m a i n s o f t h e r e c e p t o r m o d e l h a s 18 n e g a t i v e l y c h a r g e d r e s i d u e s ( A s p a n d G l u ) a n d 9 p o s i t i v e l y c h a r g e d r e s i d u e s (Lys, A r g a n d H i s ) . T h e c y t o p l a s m i c d o m a i n s w h i c h w e r e inc l u d e d in t h e m o d e l h a d 17 n e g a t i v e l y c h a r g e d r e s i d u e s a n d 29 p o s i t i v e l y c h a r g e d r e s i d u e s . T h e t r u n c a t e d fragment of the cytoplasmic loop between helix 5 and helix

F

I

CA CD

J

~CD

I

I

CD

CD CA

C5

E ~ N

\

6

c3

/

/ /

\

C2~C2

/

O Fig. 1. Atom types of ritanserin in the united atom model.

c2--

-cz

/

I CZ

\

co

\

/ \ CD ~

NF

170 6 had 3 negatively charged residues and 8 positively charged residues. The transmembrane parts of the receptor model had five charged amino acids: A s p 120 in helix 2, Asp 155 in helix 3, His 165 one helical turn from the cytoplasmic end of helix 3, Lys 191 and Lys 195. both near the cytoplasmic end of helix 4. The electrostatic potentials around the receptor model ranged from -51 kcal/mol to +45 kcal/mol and were mainly negative on the synaptic side, as shown in Fig 2. Lowest negative electrostatic potentials were found around Asp 232 near the synaptic opening of the central duct. The cytoplasmic domains were dominated by positive potentials, as shoran in Fig. 2. The C-terminal tail has 11 positively charged and 10 negatively charged amino acids. However, 9 out of these I0 negatively

charged residues are located among the 40 residues at the C-terminal end, and the negative electrostatic potentials on the cytoplasmic side were mainly distributed around these 40 residues. The area around Asp 12(I and Asp 155 in the central core of the receptor model, had negative electrostatic potentials ranging from - 5 0 to -15 kcal/mol. Ser 159 was located between Asp 120 and Asp 155, and enforced the negative electrostatic potentials in this area of the putative ligand binding site.

5-Hf-receptor interactions The 20 ps molecular dynamics simulation (A) of 5-HT in a static receptor model was started with the ligand between the N-terminal and the synaptic opening of the

Fig. 2. Initial energy minimized receptor model. Left part: chain of C,, carbon atoms. Red, positively charged amino acids (Lys, Arg, His): blue, negatively charged amino acids (Glu, Asp). Right part: water-accessible surface, color-coded according to electrostatic potentials 1.4 A outside the surface. Red, e > 20 kcal/mol: white, 20 /> e~ > -20 kcal/mol: blue. e < 20 kcal/mol.

171

Fig. 3 (Top). Structures observed after 20 ps (green), 80 ps (red) and 100 ps (yellow) of molecular dynamics simulation of the 5-HT 2 receptor model with 5-HT at the putative binding site. Upper: C a carbon atoms of the seven transmembrane a helices, side chain of Asp 120 in helix 2, and 5-HT. The helices are viewed from the synaptic side, and are in anti-clockwise order. Lower: detail showing helix 2 with the carboxylic side chain of Asp 120 near the amino group of 5-HT. The 5-HT molecule and the side chain of Asp 120 moved in a concerted fashion during the simulation. Fig. 4 (Bottom). Stereo view of 5-HT and side chains of interacting amino acid residues after 100 ps molecular dynamics simulation and energy minimization. Color coding: Thr 88 in helix 1, green; Asn 92 in helix 1, yellow; Asp 120 in helix 2, blue; Asp 155 in helix 3, blue; Phe 243 and Phe 244 helix 5, green; Trp 336 in helix 6, yellow. White dots show the van der Waals surface of 5-HT.

172 TABLE III Ligand-receptor interactions with amino acid residues having van der Waals contact with the ligand after 100 ps molecular dynamics simulation with 5-HT (B) or ritanserin (C and D), and energy minimization of the complex

Other G protein-coupled neurotransmitter receptors where the residue is conserved are indicated. (All = all known sequences.) Residue

Helix no.

Interaction energy (kcal.mo1-1) Ser. (B)

Thr 88 Gly 91 Ash 92

I

-5.3

I I

0.1 -7.8

Asp 120 Leu 123 Gly 124 Val 127 Met 128 Ser 131 Met 132 Ile 135

II II II II II II II II

-54.3

Trp 151 Asp 155

III III

Trp Ser Val Ser

200 203 204 207

IV IV IV IV

Phe Phe Phe Leu

24(1 243 244 247

V V V V

Phe 332 Met 335 Trp 336 Phe 339 Pile 340 Ash 343 Ala 346

VI VI VI VI VI VI VI

-3.0*

Rit. (C)

Conserved in receptor sequence Rit. (D)

5-HT c,a2a,O~2b,fl3,Di,D5 MpM 2 M~.M4.M ~ All All

-20.2

-10.8

-3.5 -2.1 -0.8

-8.2 -1.6 - 1.1 -0.6 -4.7 -3.6 -1.9 -0.5 -3.9 -20.9

5-HT~,,,fl2,M3,M4,M 5 5-HTl~,DpDs,apfll,fl2,fl3,M l ,M:,M3,M4,M ~ All

-2.3 -4.9

-2.8 -2.4 -3.3 -3.3

All All except 5-HTj, and D 3 DpD 4 All except D4,Mt,Mz,M3,Ma,M 5

-2.4 -1.7

-3.3

5-HTIa,5-HTIc All 5-HTlc,D4,6t2a,6~2b 5-HTla,5-HTI~,a L,ill ,flz,f13,H2

- 1.9 - 1.3 -2.3

-4.4

All ,~zt ,D4,H 2 5-HTl~,fll ,f12,/33,H2,MI,M2,M3,Ma,M5 All except M1,M2,M3,M4,M 5 5-HTIc,DI,D2,D3,D 5,azb,M 1,Mz,M~,Ma,M ~ 5-HTl~,c~L,a2~,a2b,H 2

-8.0 -5.1 -7.1

-1.6 -6.4 -8.6 -6.0 -0.4

All 5-HTLc All All except Hz,M I,M2,M3,M4,M ~ All except MpM2,M3,M4,M 5 5-HTI~,D l,Ds,fll ,f12,f13

* Outside van der Waals contact distance.

central core. D u r i n g the s i m u l a t i o n the 5 - H T m o l e c u l e

philic surface a r o u n d

was a t t r a c t e d to an area with n e g a t i v e electrostatic po-

g r o u p and the indole N - H g r o u p of 5-HT. A h y d r o p h o -

tentials n e a r A s p 31 and Ser 34 on the N - t e r m i n a l part,

bic p o c k e t s u r r o u n d i n g the ring system and part of the

the a m i n o g r o u p , the hydroxyl

and stayed in this r e g i o n for the rest of the simulation.

side chain of 5 - H T was c r e a t e d by Phe 243, Phe 244 and

D u r i n g the 100 ps simulation (B) with 5 - H T at the

L e u 247 in helix 5 and Trp 336 in helix 6 (Table III). A

proposed

binding site, the 5 - H T m o l e c u l e stayed

in

serine in helix 5, c o r r e s p o n d i n g to Ser 204 in the [32-

g a u c h e c o n f o r m a t i o n s with the torsion angle of the cen-

a d r e n e r g i c r e c e p t o r , was l o c a t e d a p p r o x i m a t e l y 2 helical

tral - C H 2 - C H 2- m o i e t y b e t w e e n 28 ° and 86 °. T h e m o v e m e n t s of the 5 - H T m o l e c u l e during the simulation w e r e

turns closer to the synaptic m e m b r a n e surface than A s p 120, t o o far f r o m 5 - H T to create any van der Waals

s y n c h r o n i z e d with the m o v e m e n t s of the side chain of

contact.

A s p 120, as illustrated in Fig. 3. A s s h o w n in Table III, strong electrostatic interactions w e r e o b s e r v e d b e t w e e n the carboxylic side chain of A s p 120 and the p r o t o n a t e d a m i n o g r o u p of 5-HT. T h e 5 - H T m o l e c u l e had van der Waals contact with

T h e total i n t e r a c t i o n e n e r g y b e t w e e n 5 - H T and a m i n o acid residues outside van der Waals c o n t a c t but within the 12 A cut-off radius, was - 1 4 . 9 keal/mol. A m o n g these, the interaction b e t w e e n 5 - H T and A s p c o u n t e d for - 3 . 0 kcal/mol.

155 ac-

various a m i n o acid residues in the final, e n e r g y - m i n i m i z e d c o o r d i n a t e set f r o m the simulation. A s indicated

c a m e slightly bent during s i m u l a t i o n B with 5-HT. P r o -

T h e t r a n s m e m b r a n e ~ helices, especially helix 6, be-

in Fig. 4, A s p 120, A s n 92 and T h r 88 c r e a t e d a hydro-

line residues did not c o n t r i b u t e especially to the b e n d -

173 TABLE IV Total interaction energy between receptor and ligand, and potential energy of the energy minimized receptor model, before and after 100 ps of molecular dynamics simulation with ligand Energy (kcal.mol -~) Simulation B

Ligand interaction Receptor

Simulation C

Simulation D

Before

After

Before

After

Before

After

-49.9 -6243

-93.0 -6907

-71.2 -6255

-98.7 -6897

-69.1 -6266

-103.1 -6850

ing of helices. The seven a helices moved into an oval arrangement during the initial 20 ps of the simulation, and stayed in an oval arrangement throughout the following 80 ps of the simulation, as shown in Fig. 3. The ends of helices showed,.a tendency to move out of an a helical conformation during the simulation. Ritanserin-receptor interactions The crystal structures of the 5-HT 2 receptor antagonists, ketanserin 31 and risperidon (C. De Ranter, private communication), which are structurally related to titanserin, both have an anti conformation of the central -CH2-CH2- fragment. The minimum-energy conformations of ritanserin had the following relative energies in vacuo: -gauche 0.0 kcal/mol, anti 6.0 kcal/mol, +gauche 6.6 kcal/mol. The low potential energy of the -gauche conformer was mainly caused by a close contact between the positively charged piperidine -NH moiety and the ketonic oxygen atom on the fused ring system. In the three different 105 ps molecular dynamics simulations of ritanserin in vacuo, starting from each of the minimum-energy conformations, ritanserin stayed in -gauche conformations during most of the simulations, while +gauche and anti conformers occasionally were observed in 5-10 ps intervals. Ritanserin also stayed in a -gauche-like conformation (-41 ° to -100 °) during simulation C with the receptor model. The ritanserin molecule rotated and translated, and at the end of the simulation the axis from the fused ring system to the center of the two fluorophenyl groups was almost perpendicular to the helical axes. The fused ring system was then directed towards helix 5, and the piperidine nitrogen atom was closer to Asp 120 than Asp 155. Helix 5 became fairly bent during simulation C, in order to accommodate the ligand. After energy minimization of the final coordinate set from the simulation, 11 different amino acid residues had van der Waals contact with the ligand (Table III). Strongest interactions were observed between ritanserin and Asp 120 and Asp 155. As shown in Table III, rela-

tively strong interactions were also observed between ritanserin and Trp 336 and Phe 340. Several residues had almost van der Waals contact with ritanserin, and the total interaction energy between ritanserin and residues within the 12/~ cut-off radius, but outside van der Waals contact distances, was -32.5 kcal/mol. During simulation D, which started with the fluorobenzene groups of ritanserin directed towards the synaptic end of the central core, the torsion angle of the central -CH2-CH2- bond varied from -40 ° to -93 °. The ritanserin molecule had van der Waals contact with several hydrophilic and hydrophobic amino acids during the simulation. After energy minimization of the final coordinate set, the ligand had strongest interaction with Asp 155, and also fairly strong interactions with Asp 120, Trp 336 and Phe 339 (Table III). The carboxylate side chain of Asp 155 was located near the piperidine nitrogen atom, while Asp 120 mainly interacted with the nitrogen atom (N6) next to the methyl group on the six-membered ring in ritanserin (Fig. 1). Trp 336 and Phe 339 were located on each side of the fused ring system, which also had contact with Trp 200. Met 132 was near one of the fiourophenyl rings, while no hydrophilic residue was observed in the proximity of the other fluorophenyl group. The ritanserin molecule rotated during the initial 40 ps of simulation D , ' s u c h that the fused ring system moved from near helix 4 and helix 5 to near helix 6. During the rest of the simulation, the fused ring system stayed directed towards helix 6. After energy minimization of the final coordinate set, the total interaction energy between ritanserin and amino acid residues within the 12/~ cut-off radius, but outside van der Waals contact distances, was -17.2 kcal/mol. As shown in Table IV, both the total ligand-receptor interaction energy and the potential energy of the receptor model decreased substantially during the 100 ps simulations (B, C and D) and subsequent energy refinement of the receptor-ligand complex. In all three simulations the transmembrane helices moved from the initial near

174

Fig. 5. Stereo view of the C,~ carbon atoms of the t r a n s m e m b r a n e a helices and the side chain of Asp L20 in helix 2 (blue) and of Asp 155 in helix 3 (blue), in the initial energy minimized receptor model and in the models obtained by 100 ps molecular dynamics simulation with a ligand. The helices are viewed from the synaptic side and have an anti-clockwise arrangement. A: initial energy refined receptor model. B: receptor model after simulation B with 5-HT. C: receptor model after simulation C with ritanserin. D: receptor model after simulation D with ritanserin. Color coding of ligand atoms: C and H, white: N, green: O, F and S, red. The white dots show the van der Waals surface of the ligand.

175 circular arrangement into a more oval arrangement, and became slightly tilted relative to each other, as shown in Fig. 5. It is interesting to note that such an oval arrangement of the a helices also was observed in bacteriorhodopsin, by electron microscopic methods 22. The relative positions of the seven transmembrane helices in the 5-HT 2 receptor model were most similar to the bacteriorhodopsin structure in the structures obtained by simulation and energy refinement with ritanserin at the putative binding site. After all three simulations both the tilt angles between helices and the size of the central core of the receptor were similar to those in bacteriorhodopsin 22. DISCUSSION As in our previous model of the dopamine D 2 receptor v, the cytoplasmic loop between helix 5 and helix 6 was truncated in the 5-HT 2 receptor model, due to the lack of any tertiary structural data. During simulation B with 5-HT the truncated third cytoplasmic loop moved closer to the C-terminal part, which was kept in a fixed position. Site-directed mutagenesis experiments have shown that segments of the third cytoplasmic loop and the C-terminal part, located near the cytoplasmic membrane surface, are involved in the coupling of flz-adrenergic receptors to Gs proteins 1°'2°. This may warrant further molecular modeling of these receptor domains. It has previously been proposed that the coupling of receptors to G proteins takes place by electrostatic interactions 3. The present calculations demonstrated that binding of a protonated ligand induces changes in the electrostatic potential of the 5-HT 2 receptor, both in the central core and in cytoplasmic domains near the membrane surface. It seems likely, therefore, that signal transduction between neurotransmitter receptors and G proteins may take place by a combination of ligand-induced changes in the electrostatic field of the receptor and conformational changes. It is possible that agonists may induce further conformational changes on a longer time scale than the 100 ps used in the present simulations. On the other hand, the major changes in the overall architecture of the receptor model and position of the ligands took place during the first 20-40 ps of the simulations. This indicates that 40 ps of simulation may be sufficiently long to examine receptor-ligand interaction energies in such models. Site-directed anti-peptide antibodies have been used to examine the topography of the fl2-adrenergic receptor 4°. The transmembrane segments of the f12 receptor, predicted from hydropathy indices of 14 different G protein-coupled neurotransmitter receptors 7, show a striking similarity with the results from the anti-peptide antibody

experiments 4°. This supports the postulated locations of the transmembrane a helices in the peptide chain of the 5-HT 2 receptor', which were determined from hydropathy indices of 14 receptor sequences 7. Minor differences between the results of the two methods were mainly due to the choice of a 27-residue helix length by the hydropathy index method and a 24-residue length by the antipeptide antibody method. The assumption that all transmembrane a helices are 27 residues long may not seem realistic. We feel, however, that the currently available tools are too inaccurate to determine the length of each helix more precisely, and have therefore chosen 27 residues as a reasonable length. The 11 membrane-spanning helices of the photosynthetic reaction center of R. viridis has an average length of 27 residues s. Recent studies of bacteriorhodopsin 22, the photosynthetic reaction center of R. viridis 9 and a plant light-harvesting complex 26, have shown transmembrane helices from 20 to 33 residues long, with a median length of 27 residues. The distribution of charged amino acid residues between synaptic, membrane-spanning and cytoplasmic domains of the 5-HT 2 receptor model (Fig. 2), resembles the distribution of charged residues in the photosynthetic reaction center of R. viridis 8"9. The mainly negative electrostatic potentials at the synaptic side and mainly positive electrostatic potentials at the cytoplasmic side of the 5-HT 2 receptor model (Fig. 2), resembles the distribution of electrostatic potentials in the dopamine D 2 r e c e p t o r model 7, and may well be a common feature of G protein-coupled neurotransmitter receptors. Since these receptors interact with protonated ligands, the negative electrostatic potentials on the synaptic side and at the putative ligand binding site, seem quite plausible. The mainly positive electrostatic potentials in the cytoplasmic domains are in accordance with site directed mutagenesis experiments which have suggested that membrane proteins generally are positive on the cytoplasmic side 3°. All this suggests that a function of positively charged residues may be to ensure a correct insertion of G protein-coupled receptors into the cell membrane, while a function of negatively charged residues may be to attract and interact with cationic ligands. Although proline residues are located within helix 2, 4, 5, 6 and 7 of the 5-HT2 receptor model, none of the helices were particularly bent at the proline residues. Also the M subunit of the photosynthetic reaction center of R. viridis has a proline near the middle of the third transmembrane helix 8"9, which demonstrates that a proline residue does not necessarily break a membranespanning ct helical structure. 5-HT and ritanserin stayed in a folded conformation during the simulations with the receptor model. How-

176 ever, the initial conformation and location of the ligand may have influenced the trajectories of molecular movements during the simulations. As shown in Table III, ritanserin had van der Waals contact with several residues in helix 2, other than Asp 120, after simulation D but not after simulation C. The simulations therefore do not rule out the possibility that also other conformations of 5-HT and ritanserin may bind to the 5-HT 2 receptor, and to other sites on the receptor. The side chain of 5-HT moved rapidly between different -gauche and +gauche conformations during molecular dynamics simulations in vacuo 13. During the 100 ps simulation of the 5-HT-receptor complex, the ligand stayed in gauche conformations near the previously reported energy minimum 13. This indicates that the side chain conformation of 5-HT is influenced by the environment at the binding site. The observed position of 5-HT after the 100 ps simulation, with a hydrophobic receptor surface enclosing the ring system and part of the side chain, and hydrophilic amino acids near the amino, indole N-H and hydroxyl groups, seems quite likely. It is interesting to note that apparently due to the strong electrostatic interaction between Asp 120 and 5-HT (Table III), the movements of the 5-HT molecule and the carboxylic side chain of Asp 120 during the simulation were highly correlated. The close contact with the protonated ligand neutralized the negative electrostatic potentials around Asp 120 which were seen in the receptor model without a ligand. Site directed mutagenesis experiments have suggested that Asn 318, Ser 204 and Ser 207 are involved in agonist binding to f12 recep tors35,37. Asn 318 in the f12 receptor is conserved in the 5-HT1A receptor, while the 5-HTlc receptor has a cysteine and the 5-HT 2 receptor a serine in the corresponding position. The 5-HT m receptor and the 5-HT 2 receptor, but not the 5-HTLA receptor, have a serine residue corresponding to Ser 204 in the flz-adrenergic receptor, and the 5-HTIA, 5-HTIc and 5-HT 2 receptors all have an alanine at the position corresponding to Ser 207 in the fl2-adrenergic receptor. In the final, energy minimized coordinate set from simulation B, 5-HT did not interact with the residues corresponding to Asn 318 and Ser 204 in the f12 receptor. The receptor interactions of ritanserin were not dominated by any single amino acid residue as was the case for 5-HT (Table III). During simulation C ritanserin moved towards Asp 120 and produced electrostatic interactions with Asp 120 and Asp 155, in addition to interactions with other hydrophilic and hydrophobic amino acids. In the ligand-receptor complex after simulation C, no polar residues were located in the area around the fluorine atoms in ritanserin. At the end of simulation D, ritanserin had van der

Waals contact with Asp 120, Asp 155 and several other residues in helices 2-6 (Table III). Although the conserved aspartic acid residues in helix 2 and helix 3 had the strongest interactions with the protonated ligand, the results shown in Table III clearly indicate that several other residues lining the central core also may contribute to ligand-receptor interactions. The ritanserin-receptor interaction energies were similar after the two simulations (C and D), and do not show preference for any of these two geometries of the ritanserin-receptor complex (Table IV). The 100 ps simulations clearly indicate, however, that 5-HT and the more bulky 5-HT 2 receptor antagonist, ritanserin, have a different pattern of interaction with Asp 120 and Asp 155. 5-HT, but not ritanserin, showed considerably stronger interactions with Asp 120 than with Asp 155. the latter being located closer to the synaptic opening of the central core of the receptor. In the present receptor model transmembrane segments 1-6 are located 1 to 5 amino acids closer to the N-terminal end and segment 7 is located 2 amino acids closer to the C-terminal end compared to another 5-HT 2 receptor model based on the structure of bacteriorhodopsin 22, where both 5-HT and the receptor were treated as relatively rigid entities 3~. Asp 155, Phe 243 and Trp 336, all of which are conserved among G protein-coupled neurotransmitter receptors 7, interacted with 5-HT both in the present model (Table III) and in the other 5-HT 2 receptor model 38, which had an extended conformation of 5-HT. In the other 5-HT2 receptor model 38 also Trp 151, Ser 203, Ser 207, Ser 239, Phe 339 and Phe 340 interacted with 5-HT. As shown in Table III, these residues interacted with ritanserin but not with 5-HT in the present model. In the other model the neurotransmitter was placed closer to the synaptic membrane surface, allowing the protonated amino group of 5-HT to interact with Asp 155 in helix 3 but not with Asp 120 in helix 2. The present simulation placed the neurotransmitter near the middle of the central core of the receptor, which resulted in a particularly strong interaction with Asp 120. This is in accordance with sitedirected mutagenesis experiments which have suggested that the aspartic acid residue corresponding to Asp 120 in the 5-HT2 receptor is essential for agonist binding and signal transduction in flz-adrenergic35'36, muscarinic acetylcholine M1 w and dopamine D229 receptors. The molecular dynamics simulations used in the present study both allowed the ligand to move and the receptor and ligand to adjust to each other, in a way that supposedly mimics the natural ligand binding process. The present calculations and modeling of the 5-HT 2 receptor did not take into account the possible influence of the cell membrane, water molecules and ions, on the

177 three-dimensional structure of the receptor. Phosphory-

how antagonists and agonists interact with the 5-HT 2 re-

lation and glucosylation may also affect the geometry

ceptor. The importance of the conserved aspartic resi-

and electrostatic potentials of such receptors. It should also be noted that it has not been formally proven that the seven t r a n s m e m b r a n e segments of G protein-coupled

dues in helix 2 and helix 3 for ligand binding, has been fully supported. However, our molecular modeling and calculations strongly indicate that also several other res-

neuroreceptors are a helices. Although the seven-helix

idues lining the central core of the 5 - H T : receptor may

model seems most likely, the possibility that one or sev-

interact with ligands and contribute to the specificity of ligand recognition and binding.

eral of the m e m b r a n e - s p a n n i n g segments have a fl-sheet structure, as observed in PhoE porin crystals 23, cannot be completely ruled out. In spite of these limitations, the present model and molecular dynamics simulations may shed some light on

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