3D Model for TM Region of the AT-1 Receptor in Complex with Angiotensin II Independently Validated by Site-Directed Mutagenesis Data Gregory V. Nikiforovich 1 and Garland R. Marshall Department of Biochemistry and Molecular Biophysics, Campus Box 8036, Washington University, St. Louis, Missouri 63110, Received June 29, 2001 A three-dimensional model of the complex of angio- tensin II (AII) with the transmembrane (TM) region of the angiotensin II receptor of type 1 (the AT-1 recep- tor) was obtained by molecular modeling procedures employing structural homology to the X-ray structure of rhodopsin. Since the modeling procedure consid- ered only steric and energy considerations without prior knowledge of the experimental results of site- directed mutagenesis, the results with receptor mu- tants could be used for independent validation of the model. Indeed, the model brings in contact the resi- dues of AII responsible for agonistic activity, Tyr 4 , His 6 , and Phe 8 , with many residues of AT-1 involved in signal transduction according to site-directed mu- tagenesis. The model also predicts the existence of several possible conformational pathways for trans- ferring the binding signal through the TM region of AT-1 to the intracellular loops interacting with the G-protein. © 2001 Academic Press Key Words: angiotensin II; angiotensin receptors; G-protein coupled receptors; molecular modeling. The octapeptide angiotensin II (Asp 1 -Arg 2 -Val 3 -Tyr 4 - Ile/Val 5 -His 6 -Pro 7 -Phe 8 , AII) interacts mainly with two specific receptor proteins, AT-1 and AT-2 (1). Out of these two, AT-1 is the primary vascular receptor asso- ciated with blood pressure regulation. It mediates vir- tually all of the known physiological actions of AII in cardiovascular, renal, neuronal, endocrine, hepatic, and other target cells (1). AT-1 receptors are highly homologous between various species (up to 95% (2)). AT-1 also has high homology with rhodopsin (Rh) and with the other members of the rhodopsin family of the G-protein-coupled receptors (GPCRs); see, e.g., (3). Therefore, the study of molecular mechanisms in- volved in AII–AT-1 interaction benefits rational design of new ligands of AT-1 and new mutants of that recep- tor, and also serves as a valuable prototype for the entire rhodopsin family of GPCRs. Molecular determinants of the AII–AT-1 interaction are present both in the peptide ligand as well as the transmembrane receptor. On the ligand side, many very extensive structure–activity studies with AII an- alogs (e.g., (4, 5) and references therein) showed that the moieties indispensable both for binding to the AT-1 receptor and for initiating signal transduction are the side chains of Tyr 4 , His 6 , Phe 8 , and the C-terminal carboxyl. Phe 8 is especially important for agonistic ac- tivity: a single replacement of Phe 8 for an aliphatic residue, as Ile 8 , results in an AII antagonist (4). The three-dimensional (3D) structure of the octapeptide AII has been repeatedly studied by many physicochem- ical methods including NMR, CD, IR, etc. (though not X-ray crystallography; the only exception was the com- plex of AII with an antibody (6)). Several 3D models of the “receptor-bound” conformation of AII have been suggested by molecular modeling and NMR spectros- copy of rigidified analogs of AII. We have developed one of these models previously (7, 8); this model became widely accepted in AII and AT-1 studies by other au- thors (3, 9, 10). On the receptor side, during the last several years, a variety of mutants of AT-1 have been expressed and tested for ligand binding and for inositol phosphate (IP) production (for reviews see, e.g., (9, 11)). More than 40 residues in AT-1 have been shown to be sen- sitive either to ligand binding or to signal transduction (see Table 1). In structural terms, AT-1 belongs to the so-called 7-transmembrane (7TM) proteins whose transmembrane part consists mainly of the 7-helical bundle. Very few experimental data are available on the 3D structure of AT-1. They include studies on the isolated AT-1 fragments (e.g., (12)), and some recent experimental studies employing the AII analogs with 1 To whom correspondence should be addressed. Fax: 1-314-362- 0234. E-mail: gregory@ccb.wustl.edu. Biochemical and Biophysical Research Communications 286, 1204 –1211 (2001) doi:10.1006/bbrc.2001.5526, available online at http://www.idealibrary.com on 1204 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.