However, these high-resolution structures represent static snapshots obtained under specific experimental conditions hence, they may miss important information pertinent to the conformational ensemble of GPCRs, as the receptors may have undergone a large-scale conformational transition during their (de)activation process 20, 21. Recent technological breakthroughs in structural biology, such as cryo-electron microscopy (cryo-EM) or X-ray free-electron lasers, have led to the identification of increasing GPCR structures 15, 16, 17, either in the inactive or active conformations, thereby providing mechanistic insights into the agonist-dependent receptor activation mechanisms that are useful for investigating structure-based drug design 18, 19. As an alternative strategy, targeting a binding site outside the conserved orthosteric site, also termed as an “allosteric site”, may provide avenues for the design of modulators with desirable selectivity profiles 9, 10, 11, 12, 13, 14, which is a long-standing bottleneck in GPCR drug discovery. The orthosteric site is conserved across the members of a single GPCR subfamily and, thus, poses a significant challenge in the development of selective drugs that can bind to a unique receptor subtype. In addition, some GPCRs can also transmit signals in the absence of an external stimulus or an agonist, through ‘basal’ (also known as ‘constitutive’) activity 5, 6, 7, 8.
The GPCR-mediated signal transduction is always triggered by an extracellular signal to the orthosteric site located in the extracellular region of the 7TMs bundle center, which then transduces the stimuli to the intracellular region, thereby leading to the engagement of the receptor with G proteins or β-arrestins 3, 4. These receptors share a conserved structural architecture of seven transmembrane (7TM) helices linked by three extra- (ECLs) and three intracellular loops (ICLs) 2. Together, these findings provide a deeper understanding of AT 1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT 1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.Īs the largest superfamily of cell surface proteins in the human genome, G protein-coupled receptors (GPCRs) represent the therapeutic targets of nearly one-third of all approved drugs 1. Mutation of this cryptic site prevents activation of the downstream G protein signaling and β-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Our findings suggest a synergistic transition mechanism for AT 1 receptor activation. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT 1 receptor) - a prototypical class A GPCR-activation. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs.
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