Angiotensin and Endothelin Receptor Structures With Implications for Signaling Regulation and Pharmacological Targeting

Figures

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  Figure 1. Sequence comparison between the ATRs, ETRs, and bovine rhodopsin (bOPSD) or human β-2 adrenergic receptor (hADRB2). The length of each transmembrane helix (TM1-7) or loops (IL, intracellular loop; EL, extracellular loop) are indicated above the sequence according to an AT1R structure [PDB ID: 4zud (53)] but can differ slightly in other structures. The overall sequence similarity between ETAR and ETBR is approximately 63%, whereas between AT1R and AT2R ~47%. Sequence similarities between ATRs and ETRs, respectively, are around 30%. The sequences of prototypical class A GPCRs bOPSD and hADRB2 are provided additionally for comparison. The alignment was visualized using the software BioEdit (54). Specific background colors reflect chemical properties of the amino acid side chains or the type of amino acid: black-proline; blue-positively charged; cyan/green-aromatic and hydrophobic; green- hydrophobic; red-negatively charged; gray-hydrophilic; dark red-cysteines; and magenta-histidine.

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  Figure 2. Structural features of inactive or antagonized AT1R and ETBR conformations. (A) Conserved residues in class A GPCRs (magenta sticks) important for receptor-fold, expression, and signaling are highlighted at the inactive state structure of AT1R (backbone cartoon) in complex with the antagonist ZD7155 (green sticks). Highly significant for non-active state conformations is the inward direction of the transmembrane helix (TM) 6 into the helical bundle, which closes the intracellular binding cavity for G-proteins or arrestin (see also Figure 4E ). The antagonist ZD7155 (green) is bound in a pocket between the transmembrane helices and their transition to the extracellular loops. Notably, a disulfide bridge (yellow sticks) between the N-terminus and the EL3-TM7 transition forms and stabilizes the spatial region between the N-terminus and EL3, which is also present in the AT2R and the ETBR (not shown). (B) In the antagonized ETBR structure bound with the antagonist K-8794, water molecules solved at a high resolution of 2.2 Å. These water molecules are located centrally in the helical bundle, participating by H-bonds with hydrophilic residues in maintaining an inactive state…

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  Figure 3. Thermostabilizing mutations of the ETBR and thermostabilizing mutant positions reported for class A GPCRs. (A) The five commonly and in combination used thermostabilizing ETBR substitutions are visualized at an active state structure bound with the agonist. These substitutions are also used for stabilizing inactive state and apo-state structures. (B) Positions (backbone as cyan-colored sticks) of thermostabilizing substitutions used for protein preparation of diverse class A GPCRs ( Supplementary Table S1 ) are highlighted in a rhodopsin model (only transmembrane helices), as well as (C) of thermostabilizing substitutions in determined active state structures highlighted in an opsin model (brown backbone sticks). This mapping demonstrates that substitutions contributing to thermostability can be principally designed at each transmembrane helix [see also diagram in (D)]. (D) This depiction (top view from the extracellular side) of the rhodopsin/opsin wild-type side chains of positions in class A GPCRs used for thermostabilization of both states (~130 substitutions at 97 positions, Supplementary Table S1 ) demonstrates that they act in a contrasting manner, either by modulating the…

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  Figure 4. Agonist-bound and apo-state conformations of ATRs and ETBR. (A) Diverse AT1R structures in the agonist-bound state are already available ( Table 1 ). The agonist [endogenous peptide agonist sequences are provided in (A1), including annotated disulfide bridges and conserved regions (colored background)] is bound extracellularly between the ELs and their transitions to the helices ( Figure 5 ). Several AT1R structures are stabilized intracellularly by a bound nanobody ( Table 1 ). The agonist-bound structures are not complexed yet with G-protein or arrestin. (B) The AT2R structures not only contain various agonists but have been further stabilized in some cases with Fabs (fragment antigen binding), which bind on the extracellular side. (B1) For AT2R, intracellular helix 8 has been observed to be directed inward to the transmembrane helix core and stabilizes the active state structure instead of a transducer protein like the G-protein. Generally, helix 8 is oriented parallel to the membrane and outside the helical bundle in GPCRs. (C) The active state ETBR structure bound with ET-1 represents endogenous ligand binding, whereby the ligand is buried deep within the ligand-binding…

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  Figure 5. Details of agonist binding at AT1R, AT2R, and ETBR. (A) The ligands ET and Ang (or their derivatives) are bound mainly between the N-terminus, EL2, and several transmembrane helices, whereby the ligand-binding cavity is embedded deeply toward the transmembrane helical core close to tryptophan W3666.48 (ETBR). This essential tryptophan is in direct contact with the ligand-binding site of all receptors and their endogenous peptides [also (B, B1, C)]. Of note, the arrestin-biased angiotensin II analog TRV023 does not contact the W6.48 (B-B1) (magenta translucent circle). Many hydrophilic interactions between the receptors and the peptides can be observed, whereby four positively charged lysines and three tyrosines play a fundamental role in the corresponding ligand-receptor recognition in the ETBR/ET-1 complex. Generally, EL2, EL3, and the N-terminus cover the ligand-binding pocket [(A1), inner surface representation] extracellularly for both ETBR and ATRs. Several structures reveal direct interactions between the ligand and the N-terminus, for example, in the AT1R- β-arrestin biased agonist TRV026 (6os2) and ETBR- ET-3 (6igk) complexes. (A2) The bound peptide ET-1 in the ETBR…

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  Figure 6. AT2R in complex with an antibody Fab-fragment and Ang II [PDB ID: 6jod (66)]. (A) The receptor is presented as a backbone cartoon, the ligand and the Fab are visualized with surfaces for clarity reasons. Amino acids involved in ligand and Fab binding are shown as sticks. Extracellular loops are colored red. (B) Ang II is bound deep within the helical bundle. Significant interactions can be observed with helices TM2, TM5, TM6, and EL2 (M197 backbone, R182). EL2 is involved simultaneously in Fab binding, namely with residues located in the central EL2 (E188, Y189, G191 backbone). In addition, Y106 (backbone) and D109 in the receptor EL1, contacting the Fab as well as amino acids Q37, P39 in the N-terminus. R107 in the EL1 is an important stabilizer of this constellation by several H-bonds to the transition between the N-terminus and TM1. The translucent-filled squares highlight distinguishable contact regions between the receptor with Fab and the receptor with the ligand.

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  Figure 7. Dimer arrangement of the active state AT1R bound with an Ang II analog and nanobodies. (A) The complex between the Ang II analog, AT1R, and active state stabilizing nanobodies has been crystallized as a homodimer [PDB ID: 6do1 (62)]. The interface between the protomers is constituted by hydrophobic and aromatic amino acid side chain contacts at EL1 (M90, F96), TM1 (F55, intracellularly), TM2 (Y99), and helix 3 (L100). (B) In a putative scenario of a dimeric receptor arrangement with antibody binding at one protomer, the Fab fragment should also simultaneously contact the second receptor protomer. For this model, the AT2R structure (6jod), with and without a Fab, were arranged together as suggested by the AT1R homodimer.