Energy Landscapes Reveal Agonist Control of G Protein-Coupled Receptor Activation via Microswitches

Abstract

Agonist binding to G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signaling pathways. Although high-resolution structures of different receptor states are available, atomistic details of allosteric signaling across the membrane remain elusive. We calculated free energy landscapes of β₂ adrenergic receptor activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which shed new light on how microswitches govern the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of the agonist BI-167107 is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. The connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y-Y motif), which lead to a larger population of active-like states at the G protein binding site. This coupling is augmented by protonation of the strongly conserved Asp79^2.50. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected microswitches. Characterization of these can be used to understand how ligands stabilize distinct receptor states and contribute to development drugs with specific signaling properties. The developed simulation protocol can likely be transferred to other class A GPCRs.

Figure 1

Activation mechanism of GPCRs involving a series of microswitches. Binding of the agonist BI-167107 leads to an inward bulge of TM5 (quantified as the distance between Ser207^5.46 and Gly315^7.41, red spheres), which leads to a conformational change in the connector region (Ile121^3.40 and Phe282^6.44, yellow spheres). The transmembrane cavity surrounding Asp79^2.50 is dehydrated (filled green circle), and the NPxxY motif (blue cartoon) twists upon activation, leading to the Y–Y interaction (Tyr326^7.53 and Tyr219^5.58, purple spheres). TM6 moves outward to create the G protein binding site. The inactive (PDB entry 2RH1(5)) and active (PDB entry 3P0G(6)) structures are colored white and orange, respectively.

Figure 2

Five-dimensional collective variable (CV) space used to optimize the minimum free energy path identified in a data-driven manner. (a) A fully connected neural network was trained to classify configurations in active, intermediate, and inactive metastable states (clusters). Deep Taylor decomposition was then used to identify the most important input inter-residue distances for the classification decision. The top-ranked distances were used as CVs. (b) The five CVs used in this work projected onto an intracellular view of the active crystal structure (PDB entry 3P0G). The CVs corresponding to TM2–TM7, TM6–TM4, TM7–TM4, TM3–TM6, and TM6–TM5 distances defined in Table S2 are shown as purple, blue, green, yellow, and red dashed lines, respectively. The change of these distance CVs from the inactive to the active state structures is reported in nanometers.

Figure 3

Free energy landscapes are projected onto the first two CVs used to optimize the minimum free energy path. The minimum free energy pathways were optimized (a) in the absence and (b) in the presence of the bound agonist. Characteristic events occurring during activation (see Table S3 for their definition) are marked on the string. The active and inactive labels mark the regions close to the active and inactive structures (PDB entries 3P0G(6) and 2RH1, respectively).

Figure 4

Free energy landscapes projected along variables of interest highlight changes in the pairwise coupling of microswitches following binding of an agonist ligand (middle column) and protonation of conserved residue Asp79^2.50 (right column). The free energy landscapes are projected along (a–c) the TM5 bulge (distance between Ser207^5.46 and Gly315^7.41, representing the ligand binding site contraction) and the distance between Leu272^6.34 and Arg131^3.50, representing the outward movement of TM6; (d–f) the TM5 bulge (distance between Ser207^5.46 and Gly315^7.41) and the difference between the RMSD of Ile121^3.40 and Phe282^6.44 heavy atoms to the active and inactive crystal structures,^, representing the connector region ΔRMSD; (g–i) the connector region ΔRMSD and the number of water molecules within 0.8 nm of Asp79^2.50, representing the hydration of the Asp79^2.50 cavity; (j–l) the connector region ΔRMSD and the RMSD of the NPxxY motif relative to the inactive structure 2RH1; (m–o) the connector region ΔRMSD and the distance between the two tyrosines implicated in the Y–Y interaction (Y–Y motif), and (p–r) the Y–Y motif distance and the displacement of TM6. Active and inactive state regions are labeled for each variable pair. Low-free energy regions are colored red, and high-free energy regions light yellow. Free energies are reported in kilocalories per mole. See Table S2 for microswitch definitions. (s–v) Vignettes showing the conformation of the different microswitches in the active and inactive structures 3P0G (orange) and 2RH1 (white): (s) the TM5 bulge shifting Ser207^5.46 inward, (t) the connector region containing Ile121^3.40 and Phe282^6.44, (u) the Asp79^2.50 cavity, NPxxY motif, and the two tyrosines, Tyr219^5.58 and Tyr326^7.53, of the Y–Y motif, and (v) the outward movement of TM6 upon activation.

Figure 5

Sodium ions bind to three sites in a state-dependent manner. (a) The inactive β₁AR (PDB entry 4BVN(17)) and β₂AR (PDB entry 4LDE(11)) structures and a representative inactive state simulation snapshot are shown as green, white, and blue cartoons, respectively. Representative positions of sodium ions in the MD simulations are shown as blue spheres. In the insets, a comparison to the positions of sodium ions found in the crystal structures of active β₂AR (white sphere) and inactive β₁AR (white spheres) is shown. (b and c) Free energy landscapes along the TM6 displacement and the shortest distance between Asp79^2.50 and a sodium ion for the apo and holo simulations.

Figure 6

Alternative conformation of the NPxxY identified along the most probable pathway calculated with a protonated Asp79^2.50. A representative simulation snapshot of active-like states of the β₂AR with Asp79^2.50 ionized and protonated are colored orange and light orange, respectively. A representative simulation snapshot of the inactive-like state of the β₂AR is colored white. The structural comparison highlights the resemblance between an alternative conformation of the NPxxY motif (light orange) that is favored by Asp79^2.50 protonation in the simulations and observed in three crystal structures of other class A GPCRs. Crystal structures of the other GPCRs are colored green (PDB entries 6DRX, agonist-bound 5-HT_2B serotonin receptor; 3QAK, agonist-bound A_2A adenosine receptor; and 6DO1, angiotensin II type 1 receptor in an active conformation).

Figure 7

Active and inactive class A GPCR structures cluster into two distinct groups in the five-dimensional CV space used in this work. β₂AR and 10 active (stars) and inactive (triangles) structures of other class A GPCRs are projected onto the five original CVs. Details of the mapping can be found in Table S4. Their clustering into two groups highlights that the activation mechanism of all class A GPCRs can likely be described by the CVs identified in this work.