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3.3
DISCUSSION
GPCRs are allosteric proteins whose functioning fundamentals is the communication
between the two poles of the helix bundle, that is, the extracellular side receives and
transfers extracellular signals to the intracellular side deputed to recognize and ac-
tivate intracellular proteins, primarily the G protein transducers to which these recep-
tors owe their name. Activating or misfolding mutations, functionally different
ligands (e.g., agonists, biased agonists, inverse agonists, antagonists, and allosteric
modulators), and different homo/heterooligomeric states are likely to exert differen-
tial impacts on such communication.
In the last two decades or so, we paid a lot of effort in setting computational strat-
egies to infer the mechanisms of intramolecular and intermolecular communication
in a number of GPCRs of the rhodopsin family and in members of the Ras GTPase
superfamily in functionally different states (reviewed in
Fanelli & De Benedetti,
2011; Fanelli, De Benedetti, Raimondi, & Seeber, 2009; Fanelli & Raimondi,
2013
). As for GPCRs, the extensive investigations done so far led us to infer that
the main effects of activating signals, from either mutations or activating ligands,
include perturbations in the interaction pattern of the arginine in the conserved
E/DRY motif and increases in solvent exposure of selected amino acids in the neigh-
borhoods of such motif (
Fanelli & De Benedetti, 2011
). The latter effect marks the
opening of a solvent-accessible crevice between H3 and H6. These effects were sug-
gested to require the integrity of conserved amino acids in H2 and H7 (e.g., D2.50 as
well as N7.49 and Y7.53 of the NPxxY motif; here, the numbering scheme by
Ballesteros &Weinstein (1995)
is used). We also predicted that such cytosolic crev-
ice would form a receptor docking site for the C-term of the G protein
-subunit
(
Fanelli & De Benedetti, 2011
). All together, these predictions found validation
in recent advances from structure determinations, showing that the arginine of the
E/DRY motif can act both as a structural hallmark of receptor functionality and
as a recognition point for the G protein C-term (
Park, Scheerer, Hofmann, Choe, &
Ernst, 2008; Rasmussen et al., 2011; Scheerer et al., 2008
). MD simulations on a
ternary complex between agonist-bound thromboxane A2 receptor (TP) and
GDP-bound heterotrimeric Gq were the first attempt in the literature to investigate
at the atomic detail GPCR's impact on the G protein dynamics (
Raimondi, Seeber,
De Benedetti, & Fanelli, 2008
). The study suggested that the formation of a composite
receptor-G protein interface, dominated by receptor contacts with the C-term of the
a
a
-subunit, favors concerted motions of selected G protein loops in the nucleotide-
binding site and of the
a
-helical domain with respect to the Ras-like domain, features
intrinsic to the G protein structure, but amplified by receptor binding. Such interdo-
main uncoupling was related to increases in solvent exposure of GDP. In spite of
the extremely short length of simulations, the study highlighted for the first time
the displacement of the
-helical domain with respect to the Ras-like domain as
one of the early events in receptor-catalyzed GDP release. These inferences find
now support in the crystallographic complex between the
a
2AR and nucleotide-free
Gs heterotrimer (
Rasmussen et al., 2011
) and in the spectroscopic measurements
b
