Beta-2 adrenergic receptor (active state)
Beta-adrenergic receptors mediate the activation of adenylate cyclase through the action of G proteins by binding of catecholamines (such as adrenaline, dopamine). This receptor belongs to a large protein family, G-protein coupled receptor (GPCR) family. Genomes of animals contain hundreds of GPCR proteins. Beta-adrenergic receptor is regarded as a model protein of GPCR; many researchers are studying detailed molecular mechanisms of the protein.
A schematic view of GPCR molecular mechanism is shown in Figure 1. GPCR has at least two functional states; an inactive (R) state and an active (R*) state. Agonist and the heterotrimer of G proteins drive conformational equilibrium towards the active R* state. Active GPCR stimulates Galpha protein to release Gbeta and Ggamma protein and bind to adenylate cyclase. Finally it drives generation of cAMP.
In the absence of ligand molecule, GPCRs with the heterotrimer shows some basal activity. Ligands are classified according to their influence on the basal activity. “Agonist” maximally activates the activity, whereas “inverse agonist” inhibits basal activity.
Figure 1. Signal transduction mechanism of G protein coupled receptor.
The crystal structure of GPCR with inverse agonist was solved in 2007 by the collaboration of the Koblica and Stephens research groups (PDBcode:2rh1). In January 2011, the crystal structure of active GPCR with agonist was reported in Nature. This eProtS article reports the active structure, mainly describes the difference between inactive and active structures.
The intracellular loop was replaced by T4 lysozyme to crystallize the beta 2 adrenergic receptor, whereas the T4 lysosyme was not visible in the crystal structure. To stabilize the active state, agonist ligand BI-167107 was added to the receptor. Furthermore, a nanobody (functional antibody molecule without light chains) was introduced to mimic the heterotrimer of G-protein. To generate receptor-specific nanobodies, a llama was immunized with agonist-bound receptors. Finally, this PDB entry 2p0g contains three molecules; beta-2 adrenergic receptor, agonist BI-167107, and nanobody.
Difference between inactive and active conformations
Following figures compares the inactive receptor structure (PDBcode : 2rh1) and active receptor structure (PDBcode: 3p0g).
Figure 2 shows a comparison of overall structures. The largest differences are found at the cytoplasmic face of the receptor; outward movements of TM5 and TM6, and inward movements of TM3 and TM7. The largest change is observed in TM6, with an 11.4 angstrom movement. This region corresponds to the interface with nanobody. If the nanobody has a similar role to the heterotrimer of G-proteins, the interaction with the heterotrimer is essential to stabilize the active state.
Figure 2. Comparison of overall structures of inactive and active states.
Figure 3 shows a comparison of ligand-binding sites. The inactive structure (2rh1) has an inverse agonist, carazolol ( “CAU” in PDB data), whereas the active structure has an agonist, BI-167107 (“P0G” in PDB data). In contrast to the large change observed in the cytoplasmic domain of the receptor, the changes in the ligand-binding pocket are small. Most of interacting amino acids for the inverse agonist are similar to those for the agonist. The largest difference between inactive and active ligand-binding sites is observed in Ser 207 in TM5, whose Calpha position shifts by 2.1 angstrom.
Figure 3. Comparison of ligand-binding site structures of inactive and active states.
Although the differences in ligand binding site are small, these small changes may be associated with the 11 angstrom outward movement of the cytoplasmic end of transmembrane helix 6.
Protein Data Bank (PDB)
Rasmussen, S.G. , Choi, H.J. , Fung, J.J. , Pardon, E. , Casarosa, P. , Chae, P.S. , Devree, B.T. , Rosenbaum, D.M. , Thian, F.S. , Kobilka, T.S. , Schnapp, A. , Konetzki, I. , Sunahara, R.K. , Gellman, S.H. , Pautsch, A. , Steyaert, J. , Weis, W.I. , Kobilka, B.K. Structure of a nanobody-stabilized active state of the b2 adrenoceptor. Nature, (2011) 469:175-180 PubMed:21228869.
author: Takeshi Kawabata