Gating Mechanism for Biased Agonism at Angiotensin II Type 1 Receptors

Figures

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  Figure 1. Pathological processes and diseases associated with the dysregulation of AngII and overactivation of AT1R. Increased levels of AngII and subsequent overstimulation of AT1R have clinically been correlated with deleterious processes and diseases involving the cardiovascular [7,10,11,14], central nervous [15,16], endocrine [17,18,19,20], gastrointestinal [1,21], hepatic [22,23], pulmonary [24], renal [10,25], and reproductive [26,27,28] systems. Abbreviations: AngII, angiotensin II; Arg, arginine; Asp, aspartic acid; AT1R, angiotensin II type 1 receptor; Cl−, chloride; His, histidine; Ile, isoleucine; K+, potassium; Na+, sodium; NOX, nicotinamide adenine dinucleotide phosphate oxidase; Phe, phenylalanine; Pro, proline; RNS, reactive nitrogen species; ROS, reactive oxygen species; Tyr, tyrosine; Val, valine. Key: C, carbon (black); H, hydrogen (grey); N, nitrogen (blue); O, oxygen (red); ↑, increase; ↓, decrease. Figure created using Biorender.com (accessed on 1 February 2025).

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  Figure 2. Peptides and inhibitors of the classical axis of the RAS. Angiotensinogen is catalyzed by renin, an aspartic protease produced by renal juxtaglomerular cells [29], to the relatively biologically inactive decapeptide, AngI [29]. AngI is then converted to AngII by ACE, which primarily exerts its effects by associating with AT1R [7,8]. Dysregulation of the classical axis has been well established as a major contributor underlying cardiovascular and renal diseases [12,13,30]. Medications that target and inhibit components at each step in the classical cascade, including renin inhibitors, ACE inhibitors, and ARBs, have been created to restore homeostatic balance [12,13,30]. Abbreviations: ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blockers; AngI, angiotensin I; AngII; angiotensin II; Arg, arginine; Asp, aspartic acid; AT1R, angiotensin II type 1 receptor; His, histidine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; Pro, proline; RAS, renin angiotensin system; Ser, serine; Tyr, tyrosine; Val, valine. Key: C, carbon (black); H, hydrogen (grey); N, nitrogen (blue); O, oxygen (red); R, sidechain (green). Figure created using Biorender.com (accessed on 1 February…

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  Figure 3. (A) AngII structure optimized in vacuo using a UHF/RM1 semiempirical QM method (0.1 kcal/mol convergence) showing CRS proton transfer (arrows). Electrostatic charge distribution of AngII is depicted as a wire-mesh: red = negative; green = neutral; blue = positive. (B) Hydrogen-dissociation energy calculations for the three neutral CRS species in AngII: Tyr8OH0e, His6NNH0e and Phe8COOH0e (see Methods). Results indicate favorable proton transfer from Tyr hydroxyl to His imidazole to Phe carboxylate (see text for details). (C,D) Non-equilibrium steered MD (SMD) calculations (UHF/RM1; see Methods) for CRS hydrogen-bonded reaction pairs: TyrO[H]0e---NNHis−1e (C), and HisNN[H]0e---OOCPhe−1e (D). Results were in overall agreement with the H-dissociation energies calculated in B and the proposed CRS mechanism (see text for details). Abbreviations: AngII, angiotensin II; His, histidine; HisNNH0e and His6NN−1e (or NNHis−1e), protonated or non-protonated His imidazole nitrogen; Ile, isoleucine; Phe, phenylalanine; Pro, proline; SMD, steered molecular dynamics; Tyr, tyrosine; TyrOH0e or TyrO−1e, protonated or non-protonated phenol OH group of Tyr; Å, Angstrom.

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  Figure 4. X-ray crystallographic conformation of the AngII oligopeptide extracted from the AT1R G protein-coupled receptor (PDB 6OS0; [37]). AngII residue labels are colored magenta while AT1R residue labels are black. Intermolecular hydrogen bonds are indicated as thick dotted yellow lines. Atom colors: cyan = C; blue = N; gray = H; for K199: red = N, blue = H. The conformation depicts a number of putative interactions between AT1R and AngII that regulate GPCR signal transduction through the CRS pathway. For example, both Asp1281 (D281) and Asp1263 (D263) of AT1R form a stabilizing H-bonded cage-like network with R2 of AngII. The H1 and H2 hydrogen positions of H6 (green bold labels) represent equilibrium tautomeric forms of neutral H6, and experimental evidence indicates that H6 participates in the CRS by accepting a proton donated by the Y4 terminal -OH group forming tyrosinate anion. Depending on a number of possible conformational changes in AngII, Y4 may relinquish its proton to either of the H6 tautomeric forms, as indicated by the black dotted lines. It is also possible that Y4 can donate its proton directly to the –COO group of F8 by means of a direct hydrogen bond, as suggested…

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  Figure 5. Gating mechanism for biased agonism at AT1R. Tyr4OH acts as a molecular switch which can swing between two poles, depending on the relative pKa of the two poles [as determined by the interactions of Arg2 (see text)]. (A) Agonist activity is engendered by the formation of a CRS wherein His6 imidazole is activated by D281. (B) Inverse agonist activity results when Tyr4 OH forms an H-bond with the C-terminal carboxylate [in a salt bridge with K199]. The single-letter code for amino acids denotes receptor-based residues. Note that for figurative purposes aromatic rings are not shown in their true orientations (see text for details). Abbreviations: Arg, arginine; C, carbon (black); H, hydrogen (grey); H-bond, hydrogen bond; His, histidine; N, nitrogen (blue); O, oxygen (red); Phe, phenylalanine; Tyr, tyrosine. Figure created using Biorender.com (accessed on 1 February 2025).

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  Figure 6. AngII-induced contraction responses in mouse and rat iliac arteries. An AngII dose–response test caused biphasic vasoactive responses (mean ± SEM), which involve contraction responses followed by reduction contraction and relaxation. Abbreviations: AngII, angiotensin II; IL, iliac artery; SEM, standard error of mean.