Modeling Angiotensin II‐mediated activation of the Angiotensin II Type 1 Receptor

Abstract

It is well known that agonist binding to GPCRs engages a structural rearrangement, leading to GDP disassociation from the G‐alpha subunit and subsequent GTP association and activation of the Gα subunit. However, the structural mechanism mediating intramolecular molecular communication between the agonist binding pocket and G‐alpha subunit remains unknown. A meta‐analysis of 27 crystal structures of inactive and active state GPCRs demonstrated a common structural rearrangement of the GPCRs; specifically, a convergence of residue rearrangement near the G protein‐receptor interface. In an inactive state, residues 3.46 and 1.53 are in proximity to 6.37 and 7.53, respectively; whereas in an active state, a rearrangement pairs residues 5.55 and 3.46 with 6.41 and 7.53, respectively. In order to understand the structural mechanism via which Angiotensin (Ang) II induces G‐alpha activation of the Angiotensin II Type 1 Receptor (AT1R), triplicate 300 ns molecular dynamic (MD) simulations of an ‘empty’‐AT1R, constitutively active (CA) AT1R, and Ang II bound to both the wild‐type and CA AT1Rs were conducted and analyzed. After measuring RMSD of the helical residues, Ang II, and the distances of the common intra‐receptor contact residues, it was determined that the Ang II‐AT1R structures were still inactive and Ang II was still unstable. A C‐terminus Gαq fragment, containing the last 17 residues, as well as a mini‐Gαq, modeled from the mini‐Gαs crystal structure, was then inserted into the bottom of the Ang II‐AT1R complex in two separate positions (aligned and fitted) and additional MD simulations were run. RMSD of the fitted mini‐Gq systems revealed a stable structure with intra‐contact residues in mid‐transition to the active state at 300 ns of simulation. Ang II binding in each system was analyzed via MM/GBSA; additionally, the binding energy was compared to the relative stability of Ang II in the binding pocket as well as the structural changes in the AT1R to identify correlations between the binding affinity and specific structural states of Ang II within the AT1R. Additionally, common GPCR motifs: PIF (P5.50, V3.40, F6.44), DRY (D3.49, R3.40, Y3.51), and NPxxY (N7.51, P7.52, x7.53, x7.54, Y7.55) were examined in each simulation. In all Ang II‐AT1R‐Gαq models, the DRY motif salt bridge did not separate by more than 5 Å, but the PIF and NPxxY were in the active conformation states as defined by crystal structures of activated GPCRs. However, one of the triplicate MD simulation of Ang II‐CA AT1R displayed separation of the DRY with partial movement of the PIF, but no change in the NPxxY and common intra‐receptor contact residues. Taken together the data obtained thus far indicate that the simulations are in a transition state and that G proteins are required for movement of the common intra‐receptor contact residues. Further, ongoing, simulation is expected to reveal a fully active AT1R. Moreover, subsequent analysis will likely suggest the mechanism via which Ang II and Gαq binding induce structural rearrangement of the AT1R leading to an active state. Unraveling this mechanism will allow for hypothesis directed experiments to test the models and potentially elucidate novel targets to control AT1R function and understand how polymorphisms affect the AT1R.Support or Funding InformationSupport for this work is from WesternU MSPS program funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.