Tetrazole and Carboxylate Groups of Angiotensin Receptor Antagonists Bind to the Same Subsite by Different Mechanisms

doi:10.1074/jbc.270.5.2284

Tetrazole and Carboxylate Groups of Angiotensin Receptor Antagonists Bind to the Same Subsite by Different Mechanisms (*)

From the Department of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195-5069

↵^§ Present address: Dept. of Internal Medicine, Medical School of Fukuoka University, 45-1 Nanakuma Jonan-Ku, Fukuoka City, Fukuoka 814-01, Japan.
↵^¶ Present address: The Victor Chang Cardiac Research Institute, St. Vincent's Hospital, Darlinghurst, 2010 Sydney, New South Wales, Australia.

** To whom correspondence should be addressed:
Dept. of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.
Tel.: 216-444-1269; Fax: 216-444-9263.

Abstract

To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT₁ receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys and Arg mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar¹,Ile⁸]angiotensin II, and several carboxylate analogues of losartan. An Ala substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan. Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT₁ receptor involves multiple contacts with residues such as Lys and His that constitute the same subsite of the ligand binding pocket. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.

Footnotes

↵* This work was supported in part by SCOR Grant HL33713 from the National Institutes of Health, a grant-in-aid from the North East Ohio Affiliate of the American Heart Association, and a fellowship from the American Heart Association, Ohio Chapter (to K. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Z28391[GenBank].
↵¹ The abbreviations used are:

Ang II

NH₂-D-R-V-Y-I-H-P-F-COOH

Ang II-amide

NH₂-D-R-V-Y-I-H-P-F-CONH₂.
↵²S. Karnik and S.-S. Sung, unpublished observations.
↵³J. B. O. Mitchell and J. Thornton, personal communication.
- Received October 6, 1994.
- Revision received December 9, 1994.