Critical Hydrogen Bond Formation for Activation of the Angiotensin II Type 1 Receptor

G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N111 3.35 ) is such a residue within the angiotensin II type 1 (AT 1 ) receptor. Substitution of N111 3.35 for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT 1 receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D74 2.50 . Indeed, D74 2.50 forms a stable hydrogen bond (H-bond) with the residue in position 111 3.35 in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT 1 receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N46 1.50 . When expressed in HEK293 cells, the mutant N46G-AT 1 receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT 1 receptor was also inactivable. Molecular dynamics simulations also revealed the presence of a cluster of hydrophobic residues from transmembrane domains 2, 3, and 7 that appears to stabilize the inactive form of the receptor. Whereas this hydrophobic cluster and the H-bond between D74 2.50 and W111 3.35 are more stable in the inactivable N111W-AT 1 receptor, the mutant N111W/F77A-AT 1 receptor, designed to weaken the hydrophobic core, showed significant agonist-induced signaling. These results support the potential for the formation of an H-bond between residues D74 2.50 and N46 1.50 in the activation of the AT 1 receptor. Background: The N111G and N111W mutations make the AT 1 receptor constitutively active and inactivable, respectively. Results: The orientation and interactions of D74 2.50 are influenced by the residue at position 111 3.35 . Conclusion: H-bond formation between D74 2.50 and N46 1.50 is critical for AT 1 receptor activation. Significance: This novel molecular switch could be involved in the GPCR activation mechanism as it involves highly conserved residues D 2.50 and N 1.50 .