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on Gi activation by rhodopsin (
Van Eps et al., 2011
). We also speculated that the
establishment of receptor-G protein contacts is also instrumental in preventing the
establishment of intrasubunit or intersubunit interactions that would occur in
receptor-free heterotrimeric G protein. Preventing the formation of such interactions
upon receptor binding would contribute to weaken the control exerted by the
b
-subunit
on certain intradomain and interdomain motions of the
-subunit.
The representation of GPCR structures as networks of interacting amino acids
can be a meaningful way to decipher the impact of mutation, ligand binding, and/or
formation of multiprotein complex on the structural communication of the protein.
Indeed, we applied the two different variants of the PSN analysis, PSN-MD and
PSN-ENM, to investigate different aspects of GPCR function. In deep detail,
the PSN-MDmethod served to infer (a) the structural bases of rhodopsin mutations
associated with autosomal dominant retinitis pigmentosa (ADRP) (
Fanelli &
Seeber, 2010
), (b) the effect of highly conserved amino acids in the structural com-
munication of the luteinizing hormone receptor (LHR) both in its inactive and
mutation-induced active states (
Angelova et al., 2011
), and (c) the effect of ligand
binding and dimerization on the structural communication of the A
2A
adenosine
receptor (A
2A
R) (
Fanelli & Felline, 2011
). As for ADRP-linked rhodopsin muta-
tions, steered MD simulations were instrumental in simulating the unfolding pro-
cess, whereas PSN was used to infer the effects of mutations on the native
fingerprint of the hyperlinked and most stable amino acids in the structure network,
that is, native stable hubs, that oppose resistance to connectivity loss in response to
an external force (
Fanelli & Seeber, 2010
). Thus, the analysis focused on muta-
tional effects on the native stable hub frequency. The study showed that native sta-
ble hubs essentially group in the two poles of the helix bundle and along the main
axes of H3 and H6, thus suggesting that they play a role both in protein stability and
in signal transfer between extracellular and intracellular sides (
Fanelli & Seeber,
2010
). The high concentration of hubs in the retinal-binding site is consistent with
computational studies highlighting this receptor portion as a part of the stability
core and a hinge site in the dynamics of the protein (
Fanelli & Seeber, 2010;
Isin, Rader, Dhiman, Klein-Seetharaman, & Bahar, 2006; Tastan et al., 2007
). Irre-
spective of their location, misfolding mutations tend to impair selected native sta-
ble hubs in the retinal-binding site. The extent of this structural effect was found
related with the extent of the biochemical defect associated with the mutation
(
Fanelli & Seeber, 2010
). As for LHR, PSN analyses allowed the identification
of key amino acids that are part of the regulatory network responsible for propa-
gating communication between the extracellular and intracellular poles of the re-
ceptor. We found that the number of hubs and link-involving hubs in the wild type
is higher compared to the two constitutively active mutants, consistent with the
demonstrated lower stability of the active GPCR states compared to the inactive
ones (
Angelova et al., 2011
). The study emphasized the role of highly conserved
amino acids both in protein stability and in intraprotein allosteric communication.
Indeed, such amino acids behaved as stable hubs in both the inactive and active
states. Moreover, they participate as the most frequent nodes in the communication
a
