Ligand Binding Effects on Activation of Type-2 Angiotensin II Receptor

1The Angiotensin II type-2 receptor (AGTR2) is a G-protein coupled receptor (GPCR) in the Renin-Angiotensin System (RAS), mediating essential roles in cardiovascular regulation. Overactivation of AGTR2 causes to cardiovascular pathologies, ranging from hypertension to cancer. Despite its physiological importance, the molecular mechanisms driving AGTR2 activation remain poorly understood. Using long-timescale molecular dynamics (MD) simulations, we investigated the ligand binding effects of Angiotensin II (Ang II) and Angiotensin 1-7 (Ang 1-7) on AGTR2 in inactive and active states. Our results demonstrate that Ang II stabilizes the active-state of AGTR2 by reducing fluctuations in TM6 and Helix8, thereby promoting receptor activation. In contrast, Ang 1-7 modulates the inactive-state by enhancing flexibility in intracellular loops and Helix8, suggesting may a preparatory role for receptor activation. Key interactions, including hydrogen bonds (e.g., PRO7-THR1253.33 for Ang 1-7 and PHE8-LYS2155.42 for Ang II), salt bridges (e.g., ARG2-ASP2796.58 and ARG2-ASP2977.32), and hydrophobic contacts (e.g., TRP1002.60, MET1283.36), were identified as critical for receptor stability and ligand-specific dynamics. Analyses of conserved micro-switch motifs (CWxP, PIF, E/DRY, NPxxY) and intracellular distances revealed additional details about ligand-specific pathways contributing to AGTR2 activation and signal propagation. In addition, Dynamic Residue Interaction Network (DRIN) findings, aligned with micro-switch motif analyses, revealed that ligand binding impacts conserved regions, advancing our knowledge of AGTR2 activation mechanisms. These findings highlight the distinct yet complementary roles of Ang II and Ang 1-7 in regulating AGTR2 dynamics, offering new insights into GPCR activation and therapeutic opportunities for cardiovascular diseases. 2 STATEMENT OF SIGNIFICANCEUnderstanding the intricate molecular mechanisms underlying the activation of G-protein coupled receptors (GPCRs), such as the Angiotensin II type-2 receptor (AGTR2), is vital for advancing therapeutic approaches targeting cardiovascular diseases. This study reveals that both Ang II and Ang 1-7 ligands stably bind to inactive and active-state AGTR2 structures throughout 1 microsecond (s) long-timescale molecular dynamics (MD) simulations, influencing receptor dynamics and activation mechanisms within the protective arm of the Renin-Angiotensin System (RAS). While both ligands contribute to the stabilization of active-state conformations, Ang II demonstrates a more pronounced effect, potentially prolonging the receptors active state and enhancing intracellular signaling. These findings provide critical insights into the functional role of AGTR2 in RAS regulation and highlight its potential as a therapeutic target for conditions such as hypertension and other cardiovascular pathologies.