**6.1 Is the "active receptor" mobile or rigid?**

It is well known that crystallization rigidifies proteins and can lead to selection of an unusual conformation stabilized by "within the crystal" (non physiological) protein-protein interactions. For instance, opsin and metarhodopsin II conserve the same "opened" conformation in the crystal in the absence and presence of a transducin surrogate (Figure 8) (Altenbach *et al.*, 2008). Nevertheless, opsin – in contrast to metarhopsin II – has a very low ability to activate transducin: it is thus clear that crystallization of opsin "selected" a protein conformation with a low probability in intact membranes, stabilized through its contacts with other opsin molecules in the crystal.

X-ray diffraction studies have a tremendous impact on our perception of protein structure: they enhance the impression that proteins are rigid molecules with a well defined, stable conformation. In addition, the activity of most allosteric enzymes can explained in terms of two conformations with very different enzyme activities, stabilized by allosteric enhancers and inhibitors, respectively (Monod *et al.*, 1965). This is not an absolute rule, however: some enzymes change markedly in conformation upon substrate binding and dissociation – a phenomenon known as "induced fit" (Ma and Nussinov, 2010) and this can play an important role in enzyme regulation (Heredia *et al.*, 2006; Molnes *et al.*, 2011).

As suggested above, the ternary complex model had a tremendous impact on the way we understand GPCR activation, to the extent that the agonist-receptor-(empty) G protein complex is now described as "the" (one and only?) active receptor conformation – despite the fact that the Gα subunit conformation in the ternary complex (Figure 3) is not compatible with effectors activation. This interpretation was further supported by the initial computational mapping of conformational energy landscape of the β2-adrenergic receptor:

In contrast with (dark adapted) rhodopsin, a large intracellular binding pocket is present between the TM helices of metarhodopsin II (Choe *et al.*, 2011): the distance between the conserved arginine (R3.50) at the intracellular end of TM3 (E/DRY motif) and the conserved glutamate at the junction between the third intracellular loop and TM6 (E6.30) increases from less than 3.3Å in rhodopsin (PDB 1GZM), bathorhodopsin (PDB 2G87) and lumirhodopsin (PDB 2HPY) to >15 Å in opsin (PDB 3CAP, 3DBQ) and metarhodopsin II (PDB 3PQR, 3PXO) (Figure 8). Arginine R3.50 forms in opsin and metarhodopsin II a strong ion-dipole interaction with the conserved tyrosine, Y5.58 in TM5. The G-protein binding pocket is created by the rotation of TM 5 and 6. It is large enough to accommodate the C-terminal helix of the transducin Gα subunit or of GαS, at almost 40° from the membrane surface (Park *et al.*, 2008; Scheerer *et al.*, 2008; Choe *et al.*, 2011) (Figure 8): this movement forces the opening of the GDP

The sixth transmembrane helix (TM6) of the crystallized β1-, β2-adrenergic and adenosine A2A receptors remains very close to TM3 even in the presence of agonists (Rasmussen *et al.*, 2011a; Rosenbaum *et al.*, 2011; Warne *et al.*, 2011); and the conserved E/DRY motif arginine folds towards the cytoplasm, in the direction of the conserved TM6 glutamate: the G protein binding pocket is unavailable (Figure 8). An open, "metarhodopsin II-like" structure is achieved by β2-adrenergic receptors only in the presence of a G protein surrogate or of GS (Rasmussen *et al.*, 2011a; Rosenbaum *et al.*, 2011; Rasmussen *et al.*, 2011b): TM5 and TM6 rotate away from TM3, and the arginine side chain R3.50 toggles away from the IC loop E6.30

It is well known that crystallization rigidifies proteins and can lead to selection of an unusual conformation stabilized by "within the crystal" (non physiological) protein-protein interactions. For instance, opsin and metarhodopsin II conserve the same "opened" conformation in the crystal in the absence and presence of a transducin surrogate (Figure 8) (Altenbach *et al.*, 2008). Nevertheless, opsin – in contrast to metarhopsin II – has a very low ability to activate transducin: it is thus clear that crystallization of opsin "selected" a protein conformation with a low probability in intact membranes, stabilized through its contacts

X-ray diffraction studies have a tremendous impact on our perception of protein structure: they enhance the impression that proteins are rigid molecules with a well defined, stable conformation. In addition, the activity of most allosteric enzymes can explained in terms of two conformations with very different enzyme activities, stabilized by allosteric enhancers and inhibitors, respectively (Monod *et al.*, 1965). This is not an absolute rule, however: some enzymes change markedly in conformation upon substrate binding and dissociation – a phenomenon known as "induced fit" (Ma and Nussinov, 2010) and this can play an

As suggested above, the ternary complex model had a tremendous impact on the way we understand GPCR activation, to the extent that the agonist-receptor-(empty) G protein complex is now described as "the" (one and only?) active receptor conformation – despite the fact that the Gα subunit conformation in the ternary complex (Figure 3) is not compatible with effectors activation. This interpretation was further supported by the initial computational mapping of conformational energy landscape of the β2-adrenergic receptor:

important role in enzyme regulation (Heredia *et al.*, 2006; Molnes *et al.*, 2011).

binding pocket and release GDP from the G protein (Rasmussen *et al.*, 2011b).

towards the conserved tyrosine, Y5.38.

**6.1 Is the "active receptor" mobile or rigid?**

with other opsin molecules in the crystal.

preliminary results (Bhattacharya and Vaidehi, 2010) indeed suggested that full agonistsbound receptors switch spontaneously to a more stable, active conformation very similar to metarhodopsin II. Detailed computational mapping in the presence of water and lipid molecules of rhodopsin (Provasi and Filizola, 2010) and of the agonist-bound β2-adrenergic receptor (Niesen *et al.*, 2011) however indicate that both proteins are flexible and able to sample a large number of conformations. In the case of the β2-adrenergic receptor, reversible "shearing" movements of TM5-6 relative to TM 1-4 and 7 and "breathing" movements (opening and closing of the ligand binding pocket) have been predicted.

In the case of traditional receptors (including the β2-adrenergic receptor), GTP has a tremendous effect on agonists' recognition: agonists have a significantly lower affinity and much greater dissociation rate in the presence of GTP. This suggests that the predominant receptor conformation under "functional" conditions (in the presence of GTP) is different from the ternary complex conformation (that accumulates in the absence of GTP). I should like to suggest that most GPCRs are able to recruit G proteins while in the "closed" (low agonist affinity) conformation (Hu *et al.*, 2010), open a G protein binding pocket and force GDP release to achieve the "high affinity" (ternary complex) conformation, then return to the "closed" conformation upon GTP recognition and activated G protein release. Agonists do not only facilitate the transition between the "closed" and "opened" conformations described by X-ray diffraction, but also decrease the free energy difference between the ternary complex and uncoupled receptors, thereby stabilizing the empty G protein conformation and facilitating GDP release, GTP binding. G protein dissociation from the receptor is then necessary to complete G protein activation.
