Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human β2 adrenergic receptor

Abstract

G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β2 adrenergic receptor (β2 AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β2 AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.

Figure 1. SMFS of β₂AR Reconstituted into Liposomes Containing Phospholipids and Cholesterol

(A) Pushing the AFM stylus onto the proteoliposomes forces the unspecific attachment of the β₂AR polypeptide to the stylus. Retraction of the cantilever stretches the polypeptide attached to the AFM stylus and induces the sequential unfolding of the receptor. (B, C) Selection of force-distance (F-D) curves recorded upon N-terminal (B, top) and C-terminal (C, top) unfolding of β₂AR. Superimpositions of 103 (B, bottom) and 56 (C, bottom) F-D curves highlight their common features. Red lines represent WLC curves fitting the main force peaks with the number on top indicating the average contour lengths (in amino acids) revealed from the fits. Gray scale bars allow evaluating how frequently individual force peaks were populated. See also Supplemental Figures S1 and S2.

Figure 2. Structural Segments Stabilizing the Human β₂AR

Secondary (A) and tertiary (B) structure model of β₂AR. Each color represents a structural segment that is stabilized by inter- and intramolecular interactions. (A) Black amino acids (aa) highlight the end of the previous and the beginning of the next stable structural segment. This structural position corresponds to the mean contour length (given in brackets) revealed from the WLC fitting of reproducibly detected force peaks in Figure 1B. aa colored at less intensity give the standard deviation of the average force peak (Suppl. Table S1). In case the end/beginning of a structural segment had to be assumed to lie within the membrane or at the membrane surface opposite to the puling AFM stylus a certain number of aa were added to the contour length to structurally locate the segment (Experimental Procedures). All seven transmembrane α-helices of β₂AR are labeled H1-H7. Cytoplasmic and extracellular loops are indicated C1, C2, C3 and E1, E2, E3, respectively. H8 denotes the short C-terminal helix 8 at the cytoplasmic side. The secondary structural model (A) of C-terminal truncated β₂AR carrying a N-terminal FLAG epitope (blue) followed by a TEV protease cleavage site (green) was taken from (Rasmussen et al., 2007). The tertiary structural model (B) was taken from PDB ID 2RH1. See also Supplemental Figure S3 and Supplemental Table S1.

Figure 3. Free Energy Unfolding Barrier Describing Energetic (ΔG^‡) and Kinetic (k₀ and x_u) Parameters of Stable Structural Segments

(A) According to the Bell-Evans model (Evans, 1998, 2001), folded structures can be characterized using a simple two-state model. The native, folded structure resides in an energy valley and is separated by an energy barrier from the unfolded state. As approximated previously the surface roughness of the energy landscape of transmembrane α-helices, ε, is ≈4-6 k_BT (Janovjak et al., 2007). This roughness creates local energy minima that can stabilize functionally related conformational states of a structural segment. Thus, for a given surface roughness, a wide energy valley can host more conformational states (i.e., hosts a higher conformational variability) of a structural segment compared to a narrow energy valley. The transition state (‡) must be overcome to induce unfolding. x_u represents the distance between the folded state and the transition state, k₀ is the transition rate for crossing the energy barrier under zero force, and ΔG^‡ gives the activation energy for unfolding the segment. (B) Applying an external force F changes the thermal likelihood of reaching the top of the energy barrier. The energy profile along the reaction coordinate (pulling direction) is tilted by the mechanical energy -(Fcosθx, as indicated by the dashed line. The applied force does not change the ground-to-transition state distance x_u. θ describes the angle of the externally applied force relative to the reaction coordinate. As a result of this tilt, the energy barrier separating the folded from the unfolded state decreases and the probability of the folded structural segment to unfold increases.

Figure 4. DFS Plots Reveal Loading Rate Dependent Interactions Stabilizing β₂AR

For each stable structural segment of β₂AR the most probable unfolding force was plotted against the loading rate. DFS fits using Eq. 1 are shown for unliganded (red), alprenolol-bound (black), carazolol-bound (green), BI-bound (blue), THRX-bound (orange) and adrenalin-bound (violet) states. Values for x_u and k₀ obtained from fitting the DFS plots are given in Table 1. Error bars represent the standard error of most probable force and loading rate. See also Supplemental Figures S4 and S5.

Figure 5. Structural Segments of β₂AR Changing Properties upon Ligand-Binding

Structural segments that significantly change their energetic, kinetic and mechanical properties upon binding of BI, THRX or adrenalin (A), carazolol (B) and alprenolol (C) are highlighted (β₂AR structure PDB ID code 2RH1). Arrows denote increasing (arrow up) and decreasing (arrow down) parameters characterizing the width of the energy valley (x_u), transition rate (k₀), energy barrier (ΔG^‡), and spring constant (κ) of stable structural segments. Trends were taken from Table 1.