Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as targets for allosteric drug design

Abstract

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. 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 (AT1 receptor) — a prototypical class A GPCR—activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. 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. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.

Deciphering the Mechanism of Gilteritinib Overcoming Lorlatinib Resistance to the Double Mutant I1171N/F1174I in Anaplastic Lymphoma Kinase

“…The four systems were performed with two rounds of energy minimization as reported previously by a combination of steepest descent and conjugate gradient minimizations ( Lu et al, 2019b ; Mahalapbutr et al, 2020 ; Shi et al, 2020 ; Vatansever et al, 2020 ; Wang et al, 2021a ; Lu et al, 2021b ). Afterwards, 500 ps heating, and 1,000 ps equilibration at 300 K under the NVT ensemble were performed with all heavy atoms of protein-ligand complexes fixed by a 10 kcal/(mol Å 2 ) force constant.…”

Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain

“…However, due to the forbidding expense required to conduct such extensive simulations, the reproducibility of such work should be carefully assessed considering the lack of replications. In the meantime, integrating Markov state modeling (MSM) with MD simulations is gaining increasing popularity for the efficiency and accuracy that can be reached when interpreting biomolecular dynamics, and this combination has been proven reproducible when verifying with experimental techniques [ 22 , 39 ]. Nonetheless, it is clear that with very short trajectories, the intramolecular correlation network and also other important characteristics presented during the time-consuming conversion process would thus be uninvestigable.…”

“…To obtain a comprehensive insight into the dynamics of ROC homodimer during nucleotide shift, we employed large-scale all-atom molecular dynamics (MD) simulation to observe in silico at the atomistic level the intrinsic regulation of GTPase activation cycle of the ROC domain of LRRK2. Furthermore, by integrating our extensive MD simulations with the statistically robust Markov state models (MSMs) for interpreting configuration sampling [ 38 , 39 , 40 ], kinetically relevant states of ROCs homodimer as well as their interconversion rates during GDP/GTP exchange are accessible. Based on MSMs, our study provided for the first time the dynamic portrait of ROCs during nucleotide turnover, as characterized by the previously unreported coupling between dimerization and nucleotide-binding state, with the ROCs dimer exhibiting the “open” conformation with greater dimerization extent in the presence of GDP but switching to the “closed” structure with oligomerization tendency while in complex with GTP.…”

Delineating the activation mechanism and conformational landscape of a class B G protein-coupled receptor glucagon receptor

“…As a result of computation and algorithmic promotion, MD simulations have become an important source of complementary information in crystallography and a primary tool for mechanism research [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] . Furthermore, MD simulations in combination with Markov state models (MSM) have widely applied to explore the thermodynamics and kinetics of biomolecules [66] , [67] and to investigate a slew of biophysical problems, such as protein folding [68] , allosteric regulation [69] , and molecular mechanism of conformational transition [70] .…”