Agonist dynamics and conformational selection during microsecond simulations of the A(2A) adenosine receptor

Abstract

The G-protein-coupled receptors (GPCRs) are a ubiquitous family of signaling proteins of exceptional pharmacological importance. The recent publication of structures of several GPCRs cocrystallized with ligands of differing activity offers a unique opportunity to gain insight into their function. To that end, we performed microsecond-timescale simulations of the A(2A) adenosine receptor bound to either of two agonists, adenosine or UK432097. Our data suggest that adenosine is highly dynamic when bound to A(2A), in stark contrast to the case with UK432097. Remarkably, adenosine finds an alternate binding pose in which the ligand is inverted relative to the crystal structure, forming relatively stable interactions with helices I and II. Our observations suggest new experimental tests to validate our predictions and deepen our understanding of GPCR signaling. Overall, our data suggest an intriguing hypothesis: that the 100- to 1000-fold greater efficacy of UK432097 relative to adenosine arises because UK432097 stabilizes a much tighter neighborhood of active conformations, which manifests as a greater likelihood of G-protein activation per unit time.

Figure 1

Motion of ligands (A) adenosine and (B) UK432097 when bound to A_2A. The instantaneous CoM positions of the ligand are marked by black dots. In each figure, the ligand is shown by red sticks over the black dots and its average CoM position is indicated by an arrow. The transmembrane helices are shown in different colors: red (H1), green (H2), blue (H3), magenta (H4), cyan (H5), gray (H6), and orange (H7).

Figure 2

Structures of adenosine and UK432097. Marked atom numbers correspond to H-bonds listed in Table 2.

Figure 3

Orientational autocorrelation of (A) adenosine and (B) UK432097. Data for two independent trajectories of each ligand are shown. The first trajectory results are shown in solid circles and solid line, and the second trajectory results are shown by open circles and dashed line. The circles are obtained from 〈n(t)·n(t + Δt)〉, as defined in Materials and Methods. The lines are exponential functions of f(Δt) = a×exp( − (Δt / τ)) + b, fitted to the circles. The fitted decay constants τ for the adenosine case (A) are 78 ns (72 ns) for the solid (dashed) line, and for the UK432097 case (B) are 17 ns (28 ns) for the solid (dashed) line.

Figure 4

Ligand-residue contact time series for A_2A with (A) adenosine or (B) UK432097. A contact (heavy atom distance < 4 Å) is indicated by a thin vertical black line, and stable contacts appear as horizontal solid black bars. The residues shown in Table 2 are indicated, with residues identified by mutagenesis to be important for ligand binding indicated by ^∗, and those that form the binding site for our hypothesized inverted pose indicated by †. The vertical arrows indicate the transmembrane helices. The second trajectory results are shown in Fig. S2.

Figure 5

Number of water molecules in the ligand-binding pocket of A_2A with adenosine or UK432097 bound. The binding pocket is defined in Materials and Methods. The second trajectory results are shown in Fig. S3.

Figure 6

Isosurface of water density in the ligand-binding pocket of A_2A with (A) adenosine or (B) UK432097. The water density is averaged over 0.1–0.9 μs, and the isosurface with 0.3 of the bulk water density is drawn in green. In each figure, H6 and H7 are marked by VI and VII, respectively, the ligand is shown in red, and Asn-253 on H6 and His-278 on H7 are shown in blue. The portion of the ligand buried in isosurface is rendered with thinner lines. The second trajectory results are shown in Fig. S4.