Olfactory Receptor Proteins - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 55

is bound to an agonist show intermediate conformational states (e.g., A 2A adenosine
and neurotensin receptors, see Table 3.1 ). Indeed, except for rhodopsin in which the
covalently bound agonist (all-trans retinal) alone appears sufficient to stabilize the
active state conformation, experimental evidence has shown that the G protein is
required to fully stabilize the active state conformation of other GPCRs [ 37 ]. These
three structures shed light on the molecular changes occurring during receptor ac-
tivation.
Upon binding of the agonist, small conformational changes in the ligand-binding
extracellular side are amplified in the cytoplasmic side leading, more conspicu-
ously, to a large structural rearrangement of TMH6 (14 Å outward movement) and
a smaller outward movement and 7-residue cytoplasmic helix extension of TMH5
(see Fig. 3.3 ) [ 30 ]. According to Venkatakrishnan and coll. [ 34 ] the sequence of
events is the following: (i) agonist binding causes a small local structural change in
the Pro 5.50 that induces a distortion of TMH5, (ii) there is a relocation of TMH3 and
TMH7 (in the β2 adrenergic and A 2A adenosine receptors the agonist ligand pulls
the extracellular tips of TMH3, TMH5 and TMH7 inward, resulting in a moderate
contraction of the binding pocket - notice that for rhodopsin this is the contrary, the
volume of the binding pocket increases upon retinal isomerization [ 38 ]), (iii) there
is a rotation/translation of TMH5 and TMH6. Venkatakrishnan and coll. identified
a cluster of conserved hydrophobic/aromatic residues mostly at the TMH5-TMH6
interface (positions 3.40, 5.51, 6.44, 6.48). Rearrangements of residues of this in-
terface lead to the rotation of TMH6 near the conserved Phe 6.44 . Combined with the
strong kink of TMH6 at position 6.50 (which is a Pro in most GPCRs), this rotation
produces the observed large TMH6 movement. This region of TMH6 corresponds
to the sequence motif FxxCWxP (5.44-5.50). As mentioned above, neither Pro 5.50
nor the CWxP motif (6.47-6.50) is conserved in most OR sequences, therefore one
may wonder whether the activation mechanism is similar in these receptors. In the
rhodopsin complex there is also a change in the water mediated hydrogen bond
network near the NPxxY motif of TMH7 [ 36 ].
In the rhodopsin and β2 adrenergic complexes, the receptor interacts with the G
protein α-subunit through an interface made of residues belonging to ICL2, TMH3,
TMH5 and TMH6.
As mentioned above, for GPCRs other than rhodopsin, the binding of the agonist
is not sufficient to stabilize the activated state. A number of biophysical experiments
suggest that there exist several activated conformational states, and that different
ligands are able to shift the equilibrium between these states. Nygaard and coll.,
using NMR techniques complemented with molecular dynamics simulations, have
studied the existence of conformational states not observed in crystal structures
[ 37 ]. They propose the model shown in Fig. 3.4 to explain the GPCR activation
mechanisms. There are three types of conformational states: inactive, intermedi-
ate and activated. Each state consists of several microstates in which the receptor
adopts slightly different conformations. The probability of finding the system in a