Biology Reference
In-Depth Information
This chapter describes the application of PSN analysis to uncover the structural
communication in GPCRs. Strategies to highlight changes in structural communica-
tion upon misfolding mutations, dimerization, and activation are described.
Focus is put on the PSN-ENM strategy applied to the crystallographic structures
of rhodopsin in its inactive (dark) and signaling active (meta II (MII)) states,
highlighting clear changes in the PSN and the centrality of the chromophore in dif-
ferentiating the inactive and active states of the receptor.
3.1
MATERIALS
1.
The freely available Wordom software (
Seeber et al., 2011
) is employed for
performing both PSN-MD and PSN-ENM analyses. Postprocessing of the
Wordom output relies on a number of in-house made programs and scripts. The
latter provide numerical output and scripts for 3D output visualization by the
PyMOL software (
www.pymol.org
).
2.
ENM-NMA is carried out by Wordom (
Seeber et al., 2011
) as well. Three
different ENM approaches have been implemented, the rotation translation block
being the best performer one for PSN-ENM (
Raimondi, Felline, Seeber, et al.,
2013
).
3.
The PSN-ENMmethod is being also implemented in a web server with a friendly
graphical user interface (manuscript in preparation). The server allows the user to
easily set up the calculation, perform postprocessing analyses, and both visualize
and download numerical and 3D representations of the output.
4.
The GBSW implicit water/membrane model implemented in the CHARMM
molecular simulation software (
Brooks et al., 1983
) was used for GPCR
simulations.
5.
The GROMACS4 simulation package (
Hess, Kutzner, Van Der Spoel, &
Lindahl, 2008
) with the AMBER03 all-atoms force field (
Case et al.,
2005; Sorin & Pande, 2005
) was used for equilibrium MD simulations in
explicit water.
3.2
METHODS
3.2.1
Workflow of the PSN-MD and PSN-ENM approaches
The first step in PSN analysis consists in computing the protein structure graph
(PSG), that is, an ensemble of nodes and links (
Fig. 3.1
). PSG can be computed either
on an MD trajectory (PSN-MD approach) or on a single high-resolution structural
model (PSN-ENM approach). This step provides the basis to search for the shortest
paths between pairs of nodes, that is, linked nodes connecting two extremities. In this
framework, once the two extremities of interest have been specified, the algorithm
first defines all possible shortest communication paths between such extremities and
then it filters the results according to the cross correlation of atomic motions derived
