Figure 1.
Ribbon representation of the 3D structures used for the bioinformatics analysis and MD simulations.
Ribbon representation of the crystal structures of p53_human (A), p53_mouse (B) and p53_worm (C) and of the model structures of p63_human (D), p73_human (E), p53_chicken (F) and p53_fly (G). L1, L2 and L3 loops are highlighted in orange, yellow and lime, respectively. The zinc ion is represented as a purple sphere.
Figure 2.
Far-UV CD spectra of p53 family of proteins (A-G) and CD thermal melting curves (H).
CD spectra of (A) p53_human (res. 94-312), (B) p53_mouse (res. 91-308), (C) p53_worm (res. 220-420), (D) p53_fly (res. 78-277), (E) p53_chicken (res. 87-278), (F) p63_human (res. 114-326), (G) p73_human (res. 104-333). Recorded at (-) 20°C, (····) 4°C, (-·-·-) 90°C, and at (----) 20°C after heating. (H) CD thermal melting curves of (-) p53_human, (-●-) p53_mouse, (-∨-) p53_worm, (-○-) p53_fly, (-♦-) p53_chicken, (-■-) p63_human, (-□-) p73_human.
Figure 3.
Multiple structure alignment of the p53 family.
The alignment shows the conservation of the functional residues involved in zinc-binding and DNA-binding. The reported dimer and dimer-dimer interfaces are poorly conserved in D. meloganaster and C. elegans. An insertion in the zone reported to include the 5’ dimer-dimer interface residues of the isolated DBD is evident between residues 82 and 89. The lysine residue from loop 1 and terminal helix involved in DNA-binding are highlighted in orange.
Figure 4.
Structural divergences between human and worm p53 in the starting configurations for the Molecular Dynamics.
(A) p53_human. Trp146 is highlighted in green and Asn131 in magenta, S1 is shown in dark grey. (B) p53_worm. Lys266 (aligned to H. sapiens Trp146) is highlighted in green and Lys251 (aligned to H. sapiens Asn131) in magenta, S1 is shown in dark grey. Glu227 (in orange) forms a salt bridge with Lys251, thus stabilizing the S1 and S3 secondary structures and is highlighted in orange. (C) p53_human. Residues involved in the 5’ dimer-dimer interface are highlighted in dark blue. (D) p53_worm. Residues involved in the 5’ dimer-dimer interface are highlighted in dark blue. Residues corresponding to the observed insertion in p53_worm are shown in cyan, and a helical structure is present, shielding the β-barrel core from the solvent. The proximity of residues Arg298 and His391 (in orange) may confer stability to this local conformation.
Figure 5.
Cα atoms RMSD of the investigated proteins (A), loops 1 (B) and 3 (C) and number of intramolecular H-bonds of the proteins (D).
Cα RMSD of (A) the proteins, of (B) L1 loop, (C) L3 loop and (D) number of intramolecular H-bonds of p53_human (black line), p63 (red line) and p73 (green line), p53_mouse (blue line), p53_chicken (cyan line), p53_fly (purple line) and p53_worm (yellow line), computed from the starting structures as a function of the simulation time.
Figure 6.
RMSF of Cα atoms of the proteins.
RMSF of Cα atoms of p53_human (black line), p63 (red line) and p73 (green line), p53_mouse (blue line), p53_chicken (cyan line), p53_fly (purple line) and p53_worm (yellow line). The residue number refers to p53_human. Secondary structure of p53_human is displayed along the sequence (bottom panel): α-helices and β-strands are shown by red rectangles and green arrows, respectively.
Figure 7.
Cartoon representation of p53_human (A, B) and of p53_worm structures (C).
Cartoon representation of (A) starting and (B) average final structures of p53_human, highlighting the formation of H-bonds involving Gln144, Trp146 and Asp228 residues (shown as licorice). The insets show the time evolution, over the entire simulation time, of the distance between the hydrogen bond-forming atoms: (A) Gln144-Trp146 and (B) Trp146-Asp228. (C) Cartoon representation of p53_worm structure, showing the formation of a salt bridge between Glu223 and Lys268 residues (shown as licorice).
Figure 8.
Interactions of the residues of helix insertion in p53_worm.
In the starting structure (A), Arg298 and His391 are in close contact, probably forming a cation-π interaction that is lost during the simulation. Glu294 is in close contact with His391 (A), forming a salt bridge that persists for quite long time (C), but that for short time is swapped for a salt bridge with Glu388 (B, D).
Figure 9.
Electrostatic surfaces and charge distribution of the proteins.
Comparison of the electrostatic surface of the starting structures of the proteins, two views are shown for each protein.