Investigation of Agonist Binding

Molecular dynamics simulations have been extensively used to study the dynamic properties of ground-state rhodopsin in the lipid bilayer (Crozier et al. 2003; Lau et al. 2007; Huber et al. 2004; Grossfield et al. 2006; Pitman et al. 2005; Schlegel et al. 2005; Cordomi and Perez 2007). The isomerization of retinal from 11-cis to all-trans was also simulated to shed light on the structural changes that lead to the lumi-rhodopsin (LUMI) state (Crozier et al. 2007; Lemaitre et al. 2005; Martinez-Mayorga et al. 2006; Saam et al. 2002; Kong and Karplus 2007), where LUMI is the first conformational

Fig. 6.4 Resultant docking of the selective antagonist SB-228357 in the ADRB-based three-dimensional (3D) model of 5-HT2C. Amino acids residues are labeled according to the Ballesteros or 5-HT2C numbering as located in TM and E1&2 (W120, R195, N204, T205, N210) loops, respectively. Intermolecular hydrogen bonds are illustrated as dashed cylinders

Fig. 6.4 Resultant docking of the selective antagonist SB-228357 in the ADRB-based three-dimensional (3D) model of 5-HT2C. Amino acids residues are labeled according to the Ballesteros or 5-HT2C numbering as located in TM and E1&2 (W120, R195, N204, T205, N210) loops, respectively. Intermolecular hydrogen bonds are illustrated as dashed cylinders state in which the retinal is found in the all-trans form. This computational method has also been used to generate a binding mode of 5-HT2C agonists in situ.

6.3.2.1 MD Simulations

Molecular dynamics consists in simulating the temperature increase in an atomic system. According to the thermodynamic rules, the provided kinetic energy induces motion of the atoms. After the system has reached a maximal total energy, it undergoes structural distortion as the conformational space is explored during the following equilibration stage. Numerical methods as the Verlet algorithm associated to molecular force fields (as previously described for energy minimization) permit the integration of Newton's equations of motion, which highlight collective atom motions in a macromolecular system. Nowadays, the commonly accepted method of carrying out MD simulations of membrane proteins is the explicit representation of the phospholipid bilayer solvated by water to provide the most optimal environment available with the current computational power (Ivanov et al. 2005; Elmore and Dougherty 2001; Xu et al. 2005a; Xu et al. 2005b). Works of Zuo et al. and Bray et al. consisted in hypothetically binding reference agonists in the presumed inactive starting structure and proceeding MD simulations during time periods long enough to equilibrate the total energy of the complex, 2 ns in a 241 POPC (palmitoyloleoyl-phosphatidylcholine) bilayer and 5 ns in a POPE (palmitoylo-leoyl-phosphatidylethanolamine) bilayer, respectively, both with periodic boundary conditions. On one hand, apo-5-HT2C model of Zuo et al. was bound to an azepi-noindol structure according to a prior 3D pharmacophore study from 18 derivative compounds of azepinoindol structures published as 5-HT2C agonists with high affinities (Ennis et al. 2003). The deduced 3D pharmacophore is a four-point fingerprint with three aromatic and one hydrogen bond donor/acceptor features. The possible binding site for this agonist was identified on the extracellular side of the TM domain and partially covered by EL2 loop through docking of the pharmacophore elements and taking into account some published data about the position of the agonist binding site (Choudhary et al. 1993; Roth et al. 1997; Rashid et al. 2003; Kroeze et al. 2002; Kristiansen and Dahl 1996; Quirk et al. 2001; Roth et al. 1998; Wang et al. 1993). On the other hand, starting structure for the study of agonist binding by Bray et al. was a 5-HT2C-serotonin complex derived from the same sampled docking as previously described for antagonist binding.

6.3.2.2 Analysis of 5-HT2C-Agonists Interactions

As shown in Table 6.5, the main interactions observed in starting structures of 5-HT2C-agonist complex come from the conserved patch of aromatic and acidic residues widely evoked in the preceding section. Nevertheless, some key interactions observed in the "active" conformation after MD simulations significantly change. Thus, the docking of all 18 agonists of the azepinoindol family in this

Table 6.5 Interactions of 5-HT agonists

Azepinoindol derivative (Zuo's model)

Serotonin (Bray's model)

Interactions of 5-HT2C agonist

Starting complex

Final complex

Starting complex

Final complex

E2 TM5

Ile3.29(131)

Asp3.32(134)

Val3.33(135)

Ser3.36(138)

Ser3.39(141)

Val202Asn210

Val5.39(215)

Ser5.43(219)

Phe5.47(223)

Trp6.48(324)

Phe6.51(327)

Phe6.52(328)

Asn6.55(331)

Ile6.56(332)

Val7.39(354)

Tyr7.48(358)

Ionic

Aromatic

Aromatic Aromatic

VdW H bond

VdW Ionic VdW H bond

VdW VdW VdW Aromatic

Aromatic Aromatic VdW

H bond

Ionic

H bond H bond

H bond Aromatic H bond

Aromatic

Ionic H bond H bond

Aromatic H bond

Aromatic H bond

"active" conformation results in a hydrogen-bond network more tightly packed between the protonated nitrogen and Asp3.32, Ser3.36, Asn6.55, the more represented Tyr7.48 in all-azepinoindol docking, and one residue of E2 loop, namely, Asn210. In the same manner, both Asp3.32 and Ser3.36 maintain their polar interactions with the protonated amine group of serotonin as well as waters that enter the binding site and accumulate around the salt bridge between Asp3.32 and serotonin. However, the hydrogen bond with Ser5.43 and the VdW interactions with Ile6.56 are lost as the serotonin moves toward the intracellular end of the TM bundle. The hydrogen bond with Ser3.39 is lost as the hydroxyl of serotonin becomes involved in two other hydrogen bonds, as a strong hydrogen-bond acceptor for the indole of Trp6.48 and a hydrogen-bond donor for the backbone oxygen of Ser3.36.

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