Ligand-induced modulation of the free-energy landscape of G protein-coupled receptors explored by adaptive biasing techniques

Abstract

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally.

Figure 1. Free-energy of the unliganded receptor.

(A) Free-energy as a function of the position (s) along the activation pathway. Note that the curve has been shifted so that the lowest energy minima (indicated by stars) correspond to a reference free-energy value. (B) Free-energy as a function of ionic lock distance (d_IL) and the toggle switch dihedral (χ_TS) molecular switches for the unliganded receptor; contour lines are reported every k_BT.

Figure 2. Simulation results for B2AR bound to the neutral antagonist alprenolol.

(A) Free-energy profile as a function of the position (s) along the activation pathway. Note that the curve has been shifted so that the minimum (indicated by a star) corresponds to a reference free-energy value. (B) Binding mode of alprenolol. Relevant residues interacting with the ligand (any atom within a 3 Å distance cutoff) are indicated in stick representations. Helices TM5, TM6 and TM7 are shown in orange, blue and light blue respectively. Helix TM3 is shown in purple transparent representation whereas TM4 has been removed for clarity. (C) Free-energy as a function of ionic lock distance (d_IL) and the toggle switch dihedral (χ_TS) molecular switches.

Figure 3. Simulation results for B2AR bound to the inverse agonists carazolol and ICI-118,551.

(A and D) Free-energy profiles as a function of the position (s) along the activation pathway for carazolol and ICI-118,551, respectively. Note that the curves have been shifted so that the minima (indicated by stars) correspond to reference free-energy values. (B and E) Binding modes of carazolol and ICI-118,551, respectively. Relevant residues interacting with the ligands (any atom within a 3 Å distance cutoff) are indicated in stick representations. Helices TM5, TM6 and TM7 are shown in orange, blue and light blue respectively. Helix TM3 is shown in purple transparent representation whereas TM4 has been removed for clarity. (C and F) Free-energies as a function of ionic lock distance (d_IL) and the toggle switch dihedral (χ_TS) molecular switches for the carazolol- and ICI-118,551-bound B2AR, respectively.

Figure 4. Simulation results for B2AR bound to the full agonist epinephrine, the very weak partial agonist catechol, and the weak partial agonist dopamine.

(A, D, and G) Free-energy profiles as a function of the position (s) along the activation pathway for epinephrine, catechol, and dopamine, respectively. Note that the curves have been shifted so that the minima (indicated by stars) correspond to reference free-energy values. (B, E, and H) Binding modes of epinephrine, catechol, and dopamine, respectively. Relevant residues interacting with the ligands (any atom within a 3 Å distance cutoff) are indicated in stick representations. Helices TM5, TM6 and TM7 are shown in orange, blue and light blue respectively. Helix TM3 is shown in purple transparent representation whereas TM4 has been removed for clarity. (C, F, and I) Free-energies as a function of ionic lock distance (d_IL) and the toggle switch dihedral (χ_TS) molecular switches for the epinephrine-, catechol-, and dopamine-bound B2AR, respectively.

Figure 5. Structural comparisons between ligand-specific B2AR conformations.

Specifically, these comparisons (viewed from the intracellular side) are between: (A) an inverse agonist (carazolol)-bound inactive state at s∼0.2 (blue color) and a partial agonist (dopamine)-stabilized intermediate conformation at s∼0.6 (orange color); (B) an inverse agonist (carazolol)-bound inactive state at s∼0.2 (blue color) and a full agonist (epinephrine)-stabilized active conformation at s∼0.9 (pink color); and (C) a partial agonist (dopamine)-bound intermediate conformation at s∼0.6 (orange color) and a full agonist (epinephrine)-stabilized active conformation at s∼0.9 (pink color). The position of the side chains of the residues involved in the ionic locks are indicated with sticks. For clarity, IL3 has been removed.