visualized using their pathways. Compared to other existing meth-
ods, our approach proved quite powerful in the identification of
allosteric hotspot residues in GPCR signaling pathway, and corre-
lated well with the existing biochemical studies. Additionally, rota-
meric microswitches, which are deemed critical for Class A GPCR
signaling, were located accurately. The strong signals for long-
range TM communications were observed only in agonist-bound
A2AAR model system. Our method provides valuable information
on both GPCR allosteric modulation and orthosteric signaling
pathway. This simple approach could not only be extended to
other proteins but also to study the allostery of several protein–-
protein or protein–DNA/RNA complexes that are of biological
and pharmaceutical interest.4 Notes
- In our protocol, we carried out MD simulations of the model
systems through a single trajectory. However, in order to avoid
incomplete sampling of larger proteins, it is preferable to use a
multicopy approach, through which a series of simulations with
different initial velocities could be performed. - Insufficient conformational sampling limits the application of
traditional all-atom MD simulation technique. However, there
are several enhanced MD simulation techniques (Table2) avail-
able to address sampling problems and generate more diverse
structural ensembles. - An exemplary graph in Fig.2 nicely represented the signifi-
cance of betweenness centrality. The nodex has a greater
connectivity (CD¼6) to other nodes, however, the removal
ofxfrom the network does not destroy the communication
among other nodes. In contrast, removal of nodey, which has
less connectivity (CD¼4) thanx, would split the whole net-
work into three pieces. With respect to communication or the
flow of information, nodeycould be the most critical one.
Nodeyhas the highest betweenness centrality which evaluates
the importance of each node based on the amount of traffic or
the amount of internode communication. Betweenness could
be one of the most ideal measures to identify allosteric hotspots
for a given protein structure. - In our analysis, there was no significant correlation between the
residues’ centrality values in the network and sequence conser-
vation. The key residues identified from the network analysis
method were not necessarily identical to the entire set of well-
conserved residues, but to a subset of them.
466 Shaherin Basith et al.