Figure 1.
Network structure of the phototransduction model in a rod cell used in the present work.
The different forms depict the molecular species involved in the cascade, whereas lines or arrows indicate reversible or irreversible reactions, respectively. Those reactions whose kinetic parameters were changed in this study are numbered and listed accordingly in Table 1. In this respect, red numbers indicate those reactions that were changed to properly model the NCNB pathological conditions. The molecules involved in these reactions, i.e. GαGTP, PDE, and RGS, are blue, green, and magenta, respectively. Filled red rectangles indicate the stoichiometric quantity of the specific molecule in the heteromeric complex, when higher than one. Yellow stars indicate the number of PDE-activated catalytic subunits. Hexagons indicate the molecular species that were used in atomistic simulations.
Figure 2.
Structure and primary sequence of GαGTP and GαGTP-RGS-PDEγWT.
A. The cartoon representation of GαGTP structure (PDB code: 1TND) is shown. The G protein holds a Ras-like domain and an α-helical domain. The interface between α-helical and Ras-like domain makes the nucleotide binding cleft. The α-helical domain is an orthogonal bundle of six α-helices. The Ras-like domain holds a Rossmann fold, characterized by a 3-layer(αβα) sandwich architecture due to the inversion in the order of the strands β3 and β1 as well as β1 and β4, which are adjacent to each other. The Ras-like domain is colored according to secondary structure (i.e. helices, strands, and loops are, respectively, violet, yellow and white), whereas the α-helical domain is gray. The mutation site is indicated by a cyan sphere centered on the Cα-atom. The GTP nucleotide is represented by sticks colored by atoms type. The nucleotide docks into a binding site contributed by the β1/α1, α1/β2 (αF/β2 in the Gα proteins), β3/α2, β5/α4 and β6/α5 loops. These are ultraconserved regions also called G boxes 1–5 (G1–G5, colored green). G2 is also called swI (α1/β2 loop (αF/β2 loop in the Gα proteins)), whereas G3 is part of the switch II (swII, or β3/α2 loop, plus the α2-helix). The β2/β3 hairpin in between swI and swII is also called inter-switch. The β4/α3 loop, which is not a G box, is also called swIII [19]. In Gα proteins, the two domains are connected by two loops, linker 1 or α1/αA loop and linker 2 or αF/β2 loop; the latter corresponds to swI. B. According to computational experiments [20], the strands β1 and β4 divide the conserved domain into two dynamically distinct lobes, lobe 1 (i.e. the N-terminal half of the domain, colored magenta) and lobe 2 (i.e. the C-terminal half of the domain, colored blue). C. The cartoon representation of the complex involving GαGTP (gray), RGS (i.e. the RGS domain of RGS9, amino acids 286 to 418, orange), and PDEγ (green) is shown (PDB code: 1FQJ [16]). In deep detail, the RGS domain is a bundle of nine α-helices, configured into two subdomains: an N- and a C-terminal region holding an orthogonal bundle architecture (helices α1, α2, α3, α8 and α9), and a prototypical right-handed, antiparallel four-helix bundle (helices α4, α5, α6 and α7). PDEγ (residues 46–87) comprises three short α-helices and an N-terminal loop region that originates near the C-terminus and winds over helices α1 and α2. D. The primary sequence of Gα is shown. Helices, strands, and loops are, respectively, violet, yellow, and white. The G boxes are delimited by green boxes. Black numbers on the left side of the alignment refer to the sequential numbering, whereas black numbers above the sequences indicate the beginning of a secondary structure/G-box motif. An arbitrary numbering of each residue was set, characterized by the label of the secondary structure segment followed by the amino acid position within the segment. In those cases where the G-boxes overlap with the secondary structure segment, positions refer to the G-boxes. Orange and green stars mark, respectively, RGS and PDEγ recognition sites.
Figure 3.
Flash responses from wild type, GαGTPG38D+/−, and GαGTPG38D−/− rods.
Experimental (A, B, C) versus simulated (D, E, F) responses to flashes of increasing intensities from wild type GαGTPWT+/+ (A, D), heterozygous GαGTPG38D+/− (B, E) and homozygous GαGTPG38D−/− (C, F) rods are shown. Experimental data, i.e. published in Moussaif et al. [8] and provided by Marie E. Burns, refer to mice rods exposed to flashes ranging from 5 to 97000 photons µm−2 (A and B) or from 650 to 94000 photons µm−2 (C). Simulated responses derive from the model of an amphibian rod stimulated with the same light intensities as in vitro recordings. The pathological GαGTPG38D+/− and GαGTPG38D−/− models were generated by changes in the kinetic parameters kP1, kP2 and kRGS1 as described in the text. The dissimilar species justify the time scale difference between in vitro and in silico experiments. The responses were normalized with respect to the maximum photocurrent. G. Normalized simulated light response amplitude is plotted as a function of flash strength. For comparison to in vitro data, see Figure 5B in Moussaif et al. [8]. Flash intensities are the same as in D, E and F.
Table 1.
Reactions that were changed in the background of the G38D mutation.
Figure 5.
The SASA computed on Y254 (in the α3/β5 loop) is shown. Cartoons of the GαGTPWT (A) and GαGTPG38D (B) snapshots halfway through the simulation (frame 50000th) are shown, which are zoomed on the swII/α3 cleft, the primary recognition site for PDEγ. The Gα residues directly involved in PDEγ recognition (i.e. marked by green stars in the primary sequence in Figure 2) are shown in sticks. The atom color code is grey for carbon, blue for nitrogen, and red for oxygen. The residue Y254, on which the SASA index was computed, is green. The cyan ball in B indicates the mutation site. C. The time series of the SASA index calculated on Y254 along the 100 ns trajectories of GαGTPWT (violet) and GαGTPG38D (green) are shown.
Table 2.
in vitro and in silico flash responses.
Figure 4.
RMSF profiles and PCA projections.
A. The Cα-RMSF profiles from MD trajectories of GαGTPWT (violet) and GαGTPG38D (green) are shown. They refer to the 100000 frames constituting the 100 ns trajectory. The secondary structure elements are shown on the abscissa, following the Noel's nomenclature [19]. B, C, D, E. The Cα-atom projections along the linear combination of the ED analysis-derived principal components, which describe the essential subspace of the trajectories of GαGTPWT (B and C) and GαGTPG38D (D and E) are shown (see text for an explanation of ED). The number of PCs used was 111 for GαGTPWT and 74 for GαGTPG38D. Cα-atom displacements are highlighted by color ranges from violet to blue for GαGTPWT, and from green to blue for GαGTPG38D.
Table 3.
Network parameters.
Figure 6.
Global and coarse view of the communication pathways.
The global meta paths regarding GαGTPWT and GαGTPG38D in their free state (A and B panels, respectively) as well as in ternary complex with both RGS and PDEγ (C and D panels, respectively) are represented, colored violet and green, respectively. The width of each link is proportional to r, while the sphere diameter is proportional to the average r of the connecting link (see Methods for r definition). The α-helical and Ras-like domains are dark and light gray, respectively, the PDEγ binding site on Gα is aquamarine, RGS is orange and PDEγ is lemon-green. The mutation site is indicated by the red sphere.