Figure 1.
The primary and tertiary structures of Ras.
(A) The sequence of H-Ras and K-Ras (amino acid differences between K-Ras and H-Ras are indicated in cyan). Residues 12, 13, and 61, whose substitutions are associated with a large number of cancers, are colored magenta. Other residues where mutations have been reported associated with various cancers and developmental diseases, are colored in brown. Secondary structure content is indicated on top of amino acid positions with α-helices (black) and β-sheets (gray) (B) The Ras catalytic domain is composed of a six-stranded central β-sheet surrounded by five α-helices. Nucleotide binds to a conserved phosphate-binding loop (P-loop comprising residues 10–17, green) and two switch loop regions (switch 1, residues 25–40, blue, and switch 2, residues 57–75, red). Oncogenic mutations often occur at these regions, particularly residues 12, 13 and 61 [12]. The loop 3 region (orange) is also highlighted.
Figure 2.
Results of principal component analysis (PCA) on the Ras catalytic domains reveal the relationship between K-Ras and other H-Ras crystal structures.
Structures are projected onto the first three principal components (PC) with the largest variances and colored based on the dominant grouping obtained from hierarchical clustering of the projected structures. Representative structures corresponding to distinct bound-nucleotides, wild-type or mutant states, and different isoforms are labeled with their PDB IDs.
Figure 3.
Projections of MD conformers onto the first and second principal components (PC) obtained from the analysis on the Ras crystallographic ensemble.
The grey and green points represent crystallographic and MD conformers respectively.
Figure 4.
Average root mean square fluctuations (RMSF) of H-Ras, K-Ras, and H-Ras A59G from three sets of MD simulations for each system.
The standard deviations about the average are shown in gray.
Figure 5.
Dynamic cross-correlation maps (DCCMs) reveal the extent of correlation for all residue pairs in the three sets of MD simulations for the wild-type H-Ras, K-Ras, and H-Ras A59G.
Only correlation coefficients with an absolute value greater than 0.25 are displayed. Motion occurring along the same direction is represented by positive correlation (blue), whereas anti-correlated motion occurring along the opposite direction is represented by negative correlation (red).
Figure 6.
The distances between residue Q61 and γ-phosphate (black), between residue Y64 and γ-phosphate (gray) in the wild-type H-Ras, wild-type K-Ras, and H-Ras A59G.
Figure 7.
The wild-type K-Ras side chain of residue Y64 in switch 2 (red) changes orientation and moves closer toward the GTP throughout the MD simulation.
The switch 2 of 15-ns K-Ras conformation resembles the switch 2 in the wild-type H-Ras crystallographic conformation.
Figure 8.
The RMSF of GDP-bound H-Ras A59G multiple MD simulations.
(A) set 1, (B) 2, and (C) 3. Within 20-ns, only the set 2 simulation managed to reach the GDP-bound cluster of the Ras crystallographic ensemble.
Figure 9.
Spontaneous GTP-to-GDP transition in the GDP-bound H-Ras A59G MD simulation.
(A) The projection of GDP-bound H-Ras A59G MD conformers onto the first two principal components of the crystallographic ensemble. (B) The RMSDs of H-Ras A59G MD conformers with respect to the GTP-bound H-Ras A59G crystal structure (PDB 1LF0, black) and the GDP-bound H-Ras A59G crystal structure (PDB 1LF5, gray). (C) The RMSD of switch 1 (blue) and switch 2 (red) during the MD simulation.
Figure 10.
The atomic descriptions of spontaneous GTP-to-GDP transition in the H-Ras A59G MD simulation.
(A) The dynamics of secondary structures of the GDP-bound H-Ras A59G MD conformers. (B) The distances between Y32-Y40 (black), Y32-β phosphate (gray). (C) The distances between G60 and β-phosphate (black), E37-R68 (blue), E37-Y64 (yellow), E37-Y71 (green), E37-Q61 (brown) between 17.5 and 18.5 ns of MD simulation.