Lysine120 interactions with p53 response elements can allosterically direct p53 organization

Abstract

p53 can serve as a paradigm in studies aiming to figure out how allosteric perturbations in transcription factors (TFs) triggered by small changes in DNA response element (RE) sequences, can spell selectivity in co-factor recruitment. p53-REs are 20-base pair (bp) DNA segments specifying diverse functions. They may be located near the transcription start sites or thousands of bps away in the genome. Their number has been estimated to be in the thousands, and they all share a common motif. A key question is then how does the p53 protein recognize a particular p53-RE sequence among all the similar ones? Here, representative p53-REs regulating diverse functions including cell cycle arrest, DNA repair, and apoptosis were simulated in explicit solvent. Among the major interactions between p53 and its REs involving Lys120, Arg280 and Arg248, the bps interacting with Lys120 vary while the interacting partners of other residues are less so. We observe that each p53-RE quarter site sequence has a unique pattern of interactions with p53 Lys120. The allosteric, DNA sequence-induced conformational and dynamic changes of the altered Lys120 interactions are amplified by the perturbation of other p53-DNA interactions. The combined subtle RE sequence-specific allosteric effects propagate in the p53 and in the DNA. The resulting amplified allosteric effects far away are reflected in changes in the overall p53 organization and in the p53 surface topology and residue fluctuations which play key roles in selective co-factor recruitment. As such, these observations suggest how similar p53-RE sequences can spell the preferred co-factor binding, which is the key to the selective gene transactivation and consequently different functional effects.

Figure 1. Illustration of the monitored p53 core domain-REs specific interactions and p53 intra-domain interactions.

The DNA quarter-site bases are labeled as Pu1Pu2Pu3C4(A/T)5 and as Y1′Y2′Y3′G4′(T/A)5′ for the complementary chain. (A) Lys120 and Arg280 interact with the bases from the major groove while Arg248 interacts from the minor groove. Lys120 can potentially interact with bases at base positions 1–3 in a quarter site. The G bases that formed hydrogen bond with Lys120 and Arg280 are shown in thick sticks. Depending on the base identity, Lys120 may form a three-centered hydrogen bond with a G base (C) or a two-centered hydrogen bond with either a T or A base (D). Arg280 normally interacts with the G base at the 4′^th position in a quarter site that is largely conserved. Two monitored distances for Arg248 interaction with the DNA backbone are shown. (B) The salt bridge network among the base, residues Arg280, Glu281, R273 and the DNA backbone in the crystal structures is shown in dashed lines. The angle that is monitored is defined as between atoms Cα of S269, Cα of G112 and C3′ of the nucleotide at position 0 of the respective quarter site. The dihedral angle is defined by the above three atoms plus the C3′ atom at the 4′ position of the DNA. The two protein atoms are located at the centers of the well structured β-sheets and the two DNA atoms are close to the quarter site that interacted with the corresponding p53 core domain. These atoms are shown in spheres. These geometrical parameters are expected to reflect the organizational changes of p53 with respect to DNA. (C) and (D) Hydrogen bonding pattern differences between base pairs AT and GC. Hydrogen bonding donors from the DNA bases are labeled. The arrows point to the coming direction of the Lys120 or Arg280 residues from the p53.

Figure 2. Average structures of the p53-DNA complex over the last 5 ns of the Lys120 and Arg280 binding sites.

Lys120 and Arg280 are colored in cyan and the 2^nd and 4′^th bases are colored based on atom type. Hydrogen bonds formed between Lys120 and the 2^nd base or between Arg280 and the 4′^th base are shown in dotted yellow lines. The RE and its sequence for each selected structure are also listed on top of each panel. The calculations were performed with the CHARMm analysis module COOR DYNAMICS.

Figure 3. RMS deviations for residues Ly120 (black), Arg280 (red) and Arg248 (green) for each of the p53 core domains.

(A)–(F) are for REs 14_3_3σ, Gadd45, Noxa, p21, p53r2, and Puma, respectively. Calculations were performed with the CHARMm RMS module by superimposing the backbone of each p53 monomer onto the initial structure of the respective p53 monomer.

Figure 4. RMS fluctuations for each of the p53 core domain residues.

(A)–(F) are for REs 14_3_3σ, Gadd45, Noxa, p21, p53r2, and Puma, respectively. Calculations were performed with the CHARMm RMS module by superimposing the p53 backbones to illustrate the residue deviations from the initial structure. Q1, Q2, Q3 and Q4 stand for quarter sites 1, 2, 3 and 4, respectively for each of the p53-REs. Only the final 5 ns was used in the analysis.

Figure 5. Conformational changes of complex of p53 with the 14-3-3σ 1^st half site due to the change in Lys120 interaction pattern.

The cartoon representations shown in blue and green are the starting structure and the average structure over the last 5 ns, respectively. In this complex, Lys120 interacted with the 1^st G base in Q2, resulting in the shift of the p53 and affecting the organization of the other p53-quarter site interactions. In (A) and (B), the p53 core domain was superimposed for the 1^st and 2^nd quarter sites, respectively. The superimposition revealed little conformational change in p53. In (C) and (D), the DNA was superimposed for quarter sites 1 and 2, respectively. The superimposition of DNA revealed a large orientation change of p53 with respect to DNA. Structural motifs used for superposition were highlighted with the circle. (E) The structure in a different view of (C) to highlight the shift of residues Lys120 and Arg280 due to the interaction pattern change of Lys120.

Figure 6. Conformational changes of complex of p53 with the p53r2 first half site due to the change in Lys120 interaction pattern.

In Q2 of the complex, Lys120 was pushed out of the major groove and only interacted with the DNA backbone, resulting in the orientation and conformational change of p53. Coloring scheme is the same as in Fig 5. Superimposition schemes are as described in Fig 5 for panels (A), (B), (C) and (D). The superposition of the proteins shows large conformational change of p53 when Lys120 is flipped out in Q1 but the p53 structural deviation is small in Q2 when Lys120 maintains its interactions with the base. The superimposition of the DNA reveals large p53 conformational changes in both quarter sites. Structural motifs used for superposition were highlighted with the circle.

Figure 7. Selected sequences of events for correlated movements of residues Ly120 and Arg280.

(A) and (B) snapshots of conformations from the trajectory of 14-3-3σ quarter site 2 and those of p53R2 quarter site 1, respectively. Color coding of the residues are the same as in Fig 2. In 14-3-3σ quarter site 2 complex (DNA sequence is T5′G4′T3′G2′C1′), Lys120 preferred to make hydrogen bond with the G base at the 1′^st position in the complementary chain and have to move its side chain. In the p53R2 quarter site 1 complex (DNA sequence is T1G2A3C4A5), the presence of Methyl group of T base at the 1^st position destabilized the Lys120 interactions with the G base at the 2^nd position, leading to the pull-away of Lys120 from the major groove to avoid the steric clash with the Methyl group. Hydrogen bond distances were highlighted with dotted yellow lines.