Two distinct conformations of helix 6 observed in antagonist-bound structures of a beta1-adrenergic receptor

Abstract

The β(1)-adrenergic receptor (β(1)AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β(1)AR-M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R(3.50) - D/E(6.30) "ionic lock"). Here we compare eight new structures of β(1)AR-M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of β-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R(3.50)-E(6.30) distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in β(1)AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation.

Fig. 1.

Crystal contacts involving CL3. Lattice contacts between different monomers of the antiparallel crystallographic dimer are compared for different crystal forms. The thickness of the strand representing the C^α backbone reflects the temperature factor (increasing thickness reflects higher B values). Residues making crystal contacts are shown explicitly. In the case of t148, t468, t756 C2 monoclinic crystals, CL3 is in contact with CL3 of the symmetry related molecule.

Fig. 2.

Two conformations of CL3. (A) Superposition of the straight and bent H6 conformations of the carazolol-bound structures of β₁AR–M23 (crystal t1118) colored according to the B factor (increasing values from blue to red). (B) Close up of the CL3 region from panel A. (C and D) Omit densities of the CL3 for the bent and straight conformations, respectively (t1118 crystal). Densities are contoured either at (C) 0.8σ or (D) 0.7σ (gray mesh), along with the C^α trace (red).

Fig. 3.

Interhelical polar contacts at the cytoplasmic face of β₁AR–M23. (A) The structure of the bent conformation is depicted, with the intact ionic lock between Arg139 and Glu285. (B) The structure of the straight conformation is shown, with the ionic lock broken. Structures are shown as cartoons in rainbow coloration (N terminus in blue, C terminus in red), except for CL2 and the cytoplasmic extension of H6 which are in gray and residues predicted to form interhelical hydrogen bonds shown as sticks.

Fig. 4.

Sites of constitutively activating mutations (CAMs) in the CL3 region for different GPCRs. The secondary structure of the bent conformation with the ionic lock present is shown (Left) along with the CL3 region including the ends of TM5 and TM6 enlarged (Middle). The corresponding amino sequence of β36–m23 is shown for H6, with residues corresponding to CAMs in the β2 adrenergic receptor shown in yellow and the thermostabilizing mutation A282L shown in red. GPCRs with CAMs at the 6.34 site are shown on the right with reference to their evolutionary relationships. Amino acid residues corresponding to Leu289 (6.34) in the turkey β₁AR are shown in parentheses.