Identification of two distinct inactive conformations of the beta2-adrenergic receptor reconciles structural and biochemical observations

Abstract

Fully understanding the mechanisms of signaling proteins such as G protein-coupled receptors (GPCRs) will require the characterization of their conformational states and the pathways connecting those states. The recent crystal structures of the beta(2)- and beta(1)-adrenergic receptors in a nominally inactive state constituted a major advance toward this goal, but also raised new questions. Although earlier biochemical observations had suggested that these receptors possessed a set of contacts between helices 3 and 6, known as the ionic lock, which was believed to form a molecular switch for receptor activation, the crystal structures lacked these contacts. The unexpectedly broken ionic lock has raised questions about the true conformation(s) of the inactive state and the role of the ionic lock in receptor activation and signaling. To address these questions, we performed microsecond-timescale molecular dynamics simulations of the beta(2)-adrenergic receptor (beta(2)AR) in multiple wild-type and mutant forms. In wild-type simulations, the ionic lock formed reproducibly, bringing the intracellular ends of helices 3 and 6 together to adopt a conformation similar to that found in inactive rhodopsin. Our results suggest that inactive beta(2)AR exists in equilibrium between conformations with the lock formed and the lock broken, whether or not the cocrystallized ligand is present. These findings, along with the formation of several secondary structural elements in the beta(2)AR loops during our simulations, may provide a more comprehensive picture of the inactive state of the beta-adrenergic receptors, reconciling the crystal structures with biochemical studies.

Fig. 1.

Ionic lock formation in β₂AR is accompanied by transition to an inactive-rhodopsin-like conformation. (A) The β₂AR crystal structure (2RH1). (B) Close-up of the intracellular ends of helices 3 and 6 in the crystal structure showing the broken ionic lock. (C) A representative simulated conformation with the ionic lock formed. (D and E) Same conformations as shown in B and C with the homologous residues of inactive rhodopsin (1GZM; purple) superimposed. (F) C_α–C_α (light red) and N–O (light blue) distance time series for simulation c of Table 2, with smoothed versions included in dark red and blue; gray shading indicates when the smoothed C_α–C_α distance is <9.5 Å. The upper pair of gray horizontal lines indicates C_α–C_α distances of inactive rhodopsin structures 1U19 and 1L9H, and the lower pair indicates the corresponding N–O distances.

Fig. 2.

Ionic lock closure depends on the form of β₂AR. C_α–C_α and N–O distance time series for 5 simulations (simulations a and f–i in Table 2), colored as in Fig. 1F. Simulations of β₂AR-clipped and β₂AR-ICL3 (simulations a and f) exhibited C_α–C_α and N–O distances comparable to those of inactive rhodopsin most of the time; helices 3 and 6 occasionally moved apart during transient breakage of the ionic lock. By contrast, the crystallographic construct β₂AR-T4L (simulation g), and the constitutively active mutants E268A (simulation h) and D130N (simulation i), exhibited a substantially greater interhelical distance and a broken ionic lock for a larger fraction of the time.

Fig. 3.

Side chain rearrangements at the interface of helices 3, 5, and 6 enable stable ionic lock formation. In the crystal structure (gray), Y219 is located between helices 3 and 6 and contacts R131. To adopt an ionic-lock-formed conformation (yellow), Y219 rotated to the lipid-exposed side of helix 6 and L272 rotated from gauche⁻ to trans, enabling the end of helix 6 to move toward helix 3 and E268 to approach R131. Independently, Y141 rotated out from between R131 and E268 to enable formation of the R131–E268 contact.

Fig. 4.

Structural elements homologous to β₁AR form spontaneously during simulation of β₂AR. Selected side chains and adjoining helices of intracellular loop 2 (ICL2) (A) and extracellular loop 2 (ECL2) (C) of the β₂AR crystal structure. Representative helical conformation of ICL2 adopted during simulation a, superimposed on the corresponding region of the β₁AR structure (pink) (B), and of ECL2 with bound sodium ion, superimposed on the sodium binding site of β₁AR (D).