Invited Symposium: Angiotensin Receptors



Materials & Methods


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Molecular Modeling and Mutagenesis Studies of Angiotensin II/AT1 Interaction and Signal Transduction

Contact Person: Antonio C M Paiva (acmpaiva@biofis.epm.br)

Materials and Methods

Molecular modeling

To overcome the problem of comparing different receptors, a normalized numbering system was used [23]. Three-digit numbers were adopted so that the first digit (1-7) identifies the transmembrane helices, and the other two, the relative position of each residue. This procedure is adequate for comparing all classes of GPCRs since the same number (second and third digits) was attributed to the common and very conserved residues which were identified by multiple sequence alignments, as described elsewhere [1]. Before and after these attributed positions, the numbering is decreased or increased, respectively, until approaching the limits of each transmembrane region. Then, for the extramembrane regions, the process is based on other conserved positions and the absolute number values are intermediary in relation to the number of the next transmembrane region and chosen so that all sequences found in these segments for the different GPCR classes could be inserted.

For modeling the AT1 receptor the graphic program WHAT IF [24] was used to build a low-resolution molecular model for GPCRs, departing from the a-carbon coordinates described for bovine rhodopsin [25]. Alignment of receptor transmembrane sequences to the rhodopsin seven-helix bundle structure was based on functional studies obtained from mutants of all classes of receptors so that all the residues involved in retinal binding, signal transduction and G-protein coupling were set pointing towards the receptor bundle central cavity. Further, for building the AT1 receptor model, a direct transference of rhodopsin backbone coordinates was made followed by a change of residue side-chains in concordance with multiple sequence alignment of GPCRs.

For modeling the external loops of AT1, a functionally active 3D structure of AngII [26] was docked to the external region of the previously built seven-helix bundle so that the terminal carboxylate and the Arg2 side-chain of the peptide were placed at bond distances of Lys199, and of Asp278 and Asp281, respectively (for AT1 numbering see reference 27). Then the loops sequences were added so that the residues involved in peptide binding could be positioned near the AngII structure. Two disulphide bonds, between the receptor N-terminus and loop VI-VII, and between loops II-III and IV-V, were built by selecting the most usually found configurations of these bonds in PDB database which could fit the receptor-ligand structure to be modeled.

No energy minimization procedures were applied to produce an improved final form of the model but refinement commands were executed to correct structural imperfections relative to atomic distances, bond angles, backbone and side-chain dihedral angles and to debump the structures which were modeled.

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Paiva, A.C.M.; Costa-Neto, C.M.; Oliveira, L.; (1998). Molecular Modeling and Mutagenesis Studies of Angiotensin II/AT1 Interaction and Signal Transduction. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/escher/paiva0625/index.html
© 1998 Author(s) Hold Copyright