Building of the Apo5HT2C 3D Models

Classically most of the 3D homology models are built using software packages like InsightII (Accelrys Inc, San Diego, CA), Jackal (Petrey et al. 2003), or Modeller (Sali and Blundell 1993), which are able to assign the coordinates of the structurally conserved regions from the template to the model on the basis of the alignment carried ou0074 from programs like ClustalW (Thompson et al. 1994) and manually optimized. According to the multiple sequence alignment (Fig. 6.2) over the eight helices, IL1 and IL2 connecting, respectively, TM1 and TM2 then TM3 and TM4, present no gap and enough sequence conservation to be considered as structurally conserved regions in the 5-HT2C receptor. If the coordinates of conserved amino acids are inherited from those of aligned residue in the template, the coordinates of nonconserved side chains are collected from rotamer libraries derived from highresolution crystallographic structures. An ab initio method has also been employed to predict the structure of TM regions using only the amino acid sequence (Bray and Goddard 2008). As described in the preceding section, TM helical regions were predicted from hydrophobic analyses of the amino acid sequence using a program based on the Eisenberg hydrophobicity scale (Eisenberg et al. 1984). Each TM was then built into a canonical right-handed alpha helix with extended side-chain conformations and placed within a bundle according to geometric parameters of helix packing extracted from crystal structure of RHO and using a Newton-Euler inverse mass operator (NEIMO) torsional molecular dynamics (MD) method (Mathiowetz et al. 1994).

In these two methods, the variable regions such as N-terminus, EL1, EL2, and EL3 are produced separately by various algorithms that are typically based on a systematic or empirical (Monte Carlo algorithm) conformational sampling of the loop backbone followed by steps of geometry optimization using molecular mechanics methods. In this case, iterative algorithms of energy minimization are employed by applying force fields including potential energy parameters of bonded atoms (torsional, bending, and stretching energies) and nonbonded energy parameters like electrostatic and van der Waals terms. These intrinsic values are extracted from experimental crystallographic, NMR, or infrared (IR) data. Typical force fields including parameters suitable for protein structures and commonly used for the refinement of 3D homology models are chemistry at Harvard macromolecular mechanics (CHARMM) (Brooks et al. 1983) and assisted model building and energy refinement (AMBER) (Cornell et al. 1995). Modeling EL2 of 5-HT2C takes advantage of the spatial restraint caused by the disulfide bridge (conserved within the GPCR family) between Cys207, the only cystein residue of this loop, and the conserved cystein of TM3, Cys127 (C3.25). As in most cases of loops longer than eight residues, N-terminus, IL3, and C-terminus are regions that are deliberately not entirely set up in the three published models of 5-HT2C (Bray and Goddard 2008; Zuo et al. 2007; Farce et al. 2006; Rashid et al. 2003) because their higher flexibility introduces too extensive structural bias in the overall structure and can not be modeled accurately.

The resultant structures of 3D modeling undergo a procedure of geometry optimization similar to that applied for the refinement of loops. The refinement of 3D models is assisted by a theoretical checking of the helix geometry in accordance with right, allowed, or forbidden regions of the Ramachandran map (Ramachandran 1963; Ramachandran et al. 1963). This diagram is a way to visualize dihedral angles f against y of amino acid residues in protein structure. It shows the possible conformations of f and y angles for a polypeptide. In addition to the large amount of data gathered by the comparison of the models, we have produced 3D homology models (Renault et al. 2010) on the basis of the alignment with the sequence of ADRB2 according to the protocol of homology modeling just described. Providing the vicinity of sequences relative to RHO, commonly used up to now but less homologous than monoamine receptors like adrenoceptors and because no such information has been published yet, this model of the 5-HT2C receptor will be compared with published 3D models in order to reveal new structural insights.

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