Nanobody stabilization of G protein-coupled receptor conformational states

Abstract

Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.

Figure 1

Agonist-β2AR-T4L-Nb80 complex in crystals formed in lipidic cubic phase [45^••]. (a) Two different views of the crystal packing: β2AR indicated in orange and Nb80 in blue. The β2AR-nanobody complexes are arranged with the lipid bilayers approximately parallel to the bc plane of the crystal. Twofold symmetry related nanobody molecules interact along the a axis to generate a tightly packed lattice in this direction. (b) Nb80 binds to the cytoplasmic end of the β2AR, with the third complementarity determining region loop (CDR3) projecting 14Å into the core of the receptor.

Figure 2

Cytoplasmic view of the active- and inactive-state rhodopsin and β₂AR structures, compared to the antagonist and agonist bound structures of the adenosine receptor. (a) rhodopsin [6] compared to metarhodopsin II in complex with the transducing peptide [20] (b) β₂AR-TL4 bound to the inverse agonist carazolol [9] overlayed on β₂AR-TL4-Nb80 bound to the agonist BI-167107 [45^••] (c) and superposition of the antagonist ZM241385 bound [14] on the agonist UK-432097 bound adenosine receptor [49].