Serial receptor

ARBs, or sartans, are non-peptide antagonists and include the well-known anti-hypertensive drugs losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, and azilsartan, most of which share a common biphenyl-tetrazole scaffold Burnier and Brunner, ; Imaizumi et al.

These ARBs are now extensively used for the treatment of cardiovascular diseases, including hypertension, cardiac hypertrophy, arrhythmia, and heart failure. There is additional interest in ARBs regarding their efficacy in the treatment of blood-vessel diseases such as Marfan-like syndrome, aortic dissection, and aortic aneurysms Keane and Pyeritz, ; Ramanath et al.

Despite its high medical relevance and decades of research, the structure of AT 1 R and the binding mode of ARBs, however, are still unknown, which limits our understanding of the structural basis for AT 1 R function and modulation, and precludes the rational optimization of AT 1 R lead compounds. One such experimental antihypertensive compound is ZD, a high affinity antagonist and precursor to the antihypertensive drug candesartan.

ZD has a biphenyl-tetrazole scaffold similar to other ARBs, and is more potent and longer-lasting than the first clinically used ARB losartan Junggren et al. X-ray crystallography using synchrotron radiation requires sufficiently large crystals in order to collect high resolution data.

Our extensive efforts to solve the AT 1 R structure were hampered by the limited size of micro-crystals grown in the membrane mimetic matrix known as lipidic cubic phase LCP Caffrey and Cherezov, Based on the AT 1 R-ZD structure, we further performed mutagenesis and docking simulations to reveal binding modes for clinically used antihypertensive drugs targeting AT 1 R.

Eleven residues were truncated from the N-terminal region of AT 1 R Met1, Thr7-Asp16 , in order to shorten the flexible N-terminus while keeping both the putative glycosylation site at Asn4 and the disulfide bond site at Cys18 intact.

The effect of protein engineering on AT 1 R function was evaluated using radio-ligand binding and calcium mobilization assays, in which neither the truncations nor BRIL insertion significantly altered the functional and pharmacological properties of the receptor for ligand binding and signaling Figure 1B—D.

The four cysteine residues that form two disulfide bonds in the extracellular region are colored in orange. Three critical residues for ZD binding are colored in red. All other residues that interact with ZD are colored in blue.

Truncated residues are shown as light gray, and residues that do not have sufficient density in the structure and therefore were not modelled are shown in dark gray circles. AT 1 R, being the angiotensin II octapeptide receptor, shares some sequence similarity with other peptide receptors of class A GPCRs, structures of which are known sequence alignment is shown in Figure S2 , with the closest homology to the chemokine receptors e.

For example, the tilts and extensions of the extracellular ends of helices I, V, VI and VII are substantially different among these peptide receptors, while at the intracellular side, helices IV and V adopt the most diverse conformations.

The conformations of helices II and III, however, are nearly identical for all these peptide receptors. A Overall AT 1 R structure is shown as blue cartoon. ZD is shown as spheres with carbon atoms colored green. Membrane boundaries, as defined by the PPM web server Lomize et al, , are shown as planes made of gray spheres.

Intriguingly, ECL2 of AT 1 R was found to serve as an epitope for the harmful agonistic autoantibodies in preeclampsia and malignant hypertension Unal et al. Integrity of this region is also important for receptor internalization and coupling to G protein activation and signaling Thomas et al.

Experimentally, the secondary structure of AT 1 R helix VIII was observed to be sensitive to hydrophobic environment, thereby associating with the cytoplasmic side of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region Mozsolits et al.

As a separate peptide, helix VIII of AT 1 R showed a higher affinity for lipid membranes that contained negatively charged phospholipids, rather than zwitterionic phospholipids Kamimori et al. The positively charged guanidine group of Arg ECL2 forms an extensive interaction network with the acidic tetrazole and the naphthyridinone moieties of ZD Leveraging this information in mutagenesis studies, we found that mutation of Arg ECL2 to alanine abolished both the peptide and non-peptide ligands binding to AT 1 R Table S2.

However, the Arg ECL2 Lys mutant showed only 2—3 fold reduced binding affinities for ZD, which can be explained by the ability of lysine in this position to engage in salt bridge and hydrogen bond interactions similar to Arg ECL2 , although likely with less optimal interaction geometry.

An additional hydrogen bond forms between Tyr35 1. Our data showed that the Tyr35 1. Tyr 1. In the CCR5 structure, for example, Tyr37 1. A Cross-section view of AT 1 R highlighting the shape of the ligand binding pocket.

In A and B ZD is shown as sticks with yellow carbons. The residues shown by mutagenesis to be critical for ligand binding are labeled red, those that are important for either peptide or non-peptide ligands binding are labeled in yellow, and the residues that discriminate between peptide and nonpeptide ligands are labeled in purple. See also Figure S2 and Table S2. Intriguingly, some of the residues that comprise the ligand-binding pockets, including Ile 1. Residues Phe77 2.

Particularly, Trp84 2. Residues Ile31 1. Additionally, residues Val 3. In contrast, the position 3. Most of the other contacts for ZD binding to AT 1 R, however, are mediated by non-conserved residues, including Tyr87 2. These residues along with Arg ECL2 therefore define the unique shape of the AT 1 R ligand-binding pocket and explain the lack of cross-reactivity between ligands binding to AT 1 R and other peptide receptors.

The docking results show robust positioning of these compounds in the AT 1 R ligand-binding pocket Figure 4 and Table S3. Although the nature of the interactions with AT 1 R is different for each ARB given their distinct chemical structures, most of these compounds are bound in similar orientations and engage in interactions with the three residues critical for ZD binding, Arg ECL2 , Trp84 2. For example, one of the common features among these ARBs is a short alkyl tail with two-four carbons extending into a narrow hydrophobic pocket formed by Tyr35 1.

The ARBs are shown as sticks with cyan carbons. The AT 1 R residues interacting with ligands are labeled and shown as yellow lines, with the key residues highlighted in red. The hydrogen bonds are shown as black dashed lines. Pale red and pale purple dotted circles are used for groups with sub-optimal contacts as suggested by docking. The biphenyl-linker groups for hydrophobic interactions are outlined by green dashed boxes, and the two-four carbons tails, extending into the hydrophobic pocket formed by Tyr35 1.

Specific interactions of candesartan and telmisartan with Lys 5. Losartan is the first clinically used ARB for the treatment of hypertension. Docking results suggest that Arg ECL2 forms a salt bridge only with the tetrazole moiety of losartan but lacks polar interactions with other groups Figure 4 and Table S3.

Although the derived imidazole moiety of losartan can also contribute to polar interactions via methanol hydrogen bond to Cys ECL2 main chain or via nitrogen interaction with Tyr35 1.

In contrast, candesartan is an insurmountable inverse agonist with a slow dissociation rate from AT 1 R Takezako et al. The docking results indicate that besides interacting with the tetrazole moiety of candesartan, Arg ECL2 forms two salt bridges to the carboxylic group of the benzimidazole moiety Figure 4 and Table S3.

Moreover, Lys 5. Telmisartan lacks the conserved tetrazole moiety among ARBs. Instead, the carboxylic group of telmisartan is predicted to form salt bridges with both Arg ECL2 and Lys 5.

This prediction was confirmed by our mutagenesis data, which showed a dramatic decrease in affinity of telmisartan to the Tyr92 ECL1 Ala mutant Figure S3A.

Eprosartan is the most unique among the ARBs studied here, lacking both the tetrazole group and one of the two benzene rings of the biphenyl scaffold. Additionally, the specific thiophen moiety of eprosartan forms hydrophobic interactions with Pro 7. Mutation of Met 7.

On the other hand, mutations Pro 7. Finally, both our crystal structure and docking results suggest that Lys 5. Docking with the flexible side chain of Lys 5. Based on previous observations that mutations of either Asn 3. Further, it was suggested that binding of AngII to the wild-type WT receptor disrupts the hydrogen bonds between Asn 3.

Indeed, two intramolecular hydrogen bonds are observed between Asn 3. Of particular interest, Asp74 2. Moreover, the neighboring residue Phe77 2.

Combination of Phe77 2. Thus, multiple structural and functional data suggest that the hydrogen bond network around Asn 3. A A cluster of aromatic residues F77 2. The angiotensin receptor AT 1 R is a therapeutic target of outstanding interest due to its important roles in cardiovascular pathophysiology. Several AT 1 R blockers have been developed and clinically used as anti-hypertensive drugs.

Although extensive efforts were taken to delineate the pharmacophores of AT 1 R ligands, structure-based drug design was still hindered by the lack of structural information. Compared to the traditional X-ray crystallography with cryo-cooled crystals, the LCP-SFX method yields the room-temperature structure of the AT 1 R-ZD complex, which is likely to represent more accurately the receptor conformations and dynamics in the native cellular environment.

Unexpectedly, three AT 1 R residues, which have not been previously implicated in binding small molecule ligands, were found to form critical interactions with ZD; Arg ECL2 and Tyr35 1. Our extensive mutagenesis experiments revealed that residues Tyr35 1. Mutations of Ser 3. These variants may directly alter binding of ARBs and therefore modify the anti-hypertensive response to treatment with different ARBs in individuals carrying these variations.

In contrast, Leu48 1. Finally, ThrPro and ProHis are located in the C-terminal tail that was not included in the crystalized construct. Of particular interest, the atomic details of ECL2 and the extracellular ligand-binding region, revealed in the current structure, are expected to guide design of two different types of therapeutic agents targeting AT 1 R, the anti-hypertensive ARBs extensively interacting with Arg ECL2 on the ligand-binding pocket side of ECL2, and the peptide-mimicking antigens against autoantibodies, which bind to the extracellular side of ECL2 in patients with autoimmune disorders, such as preeclampsia and malignant hypertension Zhou et al, ; Fu et al, Therefore, our results provide long anticipated insights into the AT 1 R structure-function relationship and pharmacological properties, and demonstrate the potential for using the LCP-SFX method at XFEL sources to accelerate structural studies of challenging targets.

The sequence of the human AT 1 R gene was optimized for insect cells expression and synthesized by GenScript. The construct has truncations of the AT 1 R residues 1, 7—16, and — The protein was not treated with PNGase F and therefore remained fully glycosylated.

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