Crystal structure of the p53 core domain bound to a full consensus site as a self-assembled tetramer

Abstract

Recent studies suggest that p53 binds predominantly to consensus sites composed of two decameric half-sites with zero spacing in vivo. Here we report the crystal structure of the p53 core domain bound to a full consensus site as a tetramer at 2.13A resolution. Comparison with previously reported structures of p53 dimer:DNA complexes and a chemically trapped p53 tetramer:DNA complex reveals that DNA binding by the p53 core domain is a cooperative self-assembling process accompanied by structural changes of the p53 dimer and DNA. Each p53 monomer interacts with its two neighboring subunits through two different protein-protein interfaces. The DNA is largely B-form and shows no discernible bend, but the central base-pairs between the two half-sites display a significant slide. The extensive protein-protein and protein-DNA interactions explain the high cooperativity and kinetic stability of p53 binding to contiguous decameric sites and the conservation of such binding-site configuration in vivo.

Figure 1. Overall structure of the p53 core domain bound to DNA as a tetramer

(a) The tetramer viewed from the protein side. The four monomers are colored in blue (A), light green (B), light blue (C) and green (D). The same color scheme is used throughout the illustration unless indicated otherwise. The DNA is in stick model with its sequence shown below. The four pentameric motifs (quarter site) and their corresponding monomers are indicated in the sequence; (b) A view of the tetramer along the DNA axis. This view shows that the tetramer has a planer structure wherein the A-B dimer (front) and C-D dimer aling almost perfectly along the DNA axis; (c) The tetramer viewed from the DNA side. The parallelogram is shown together with the global two fold axis (dark oval) and the two local dyad aces (gray ovals). (d) A surface model of the tetramer view in the same orientation as (a). The four protein-protein interfaces are indicated.

Figure 2. Structure of the p53 core domain in the tetramer

(a) Structural variation of the L1 loop in the tetramer is shown by the comparison between monomer A (left) and monomer D (right). The electron density (sigma-a weighted, 3fo2fc, contour level at 1 e/ų) of the L1 loop of monomer D is well defined and is pointed into the major groove, whereas that of monomer A is partially disordered with a trajectory away from the major groove. This view is the same as Figure 1c; (b) The N-terminal tail has well-defined electron density. The secondary structural elements, including the zinc clusters, are indicated for both monomers (A and D). This view is the same as Figure 1a.

Figure 3. Structure of the DNA in the tetramer

The sigma-a weighted 3fo2fc density at 3 e/ų shows that the structure of the DNA is well defined except for the two ends. The DNA does not show significant bend and other major deformations from the standard B-form.

Figure 4. Conserved and variable protein-DNA interactions in the tetramer

(a) A network of interactions between β10, H2, ordered water molecules (w1, w2 and w3) and DNA is shown for monomer A (blue). This protein-DNA interaction network is also seen in monomers B, C and D (not shown) and in the p53 dimer:DNA complex (2ATA.pdb, gray) (Kitayner et al., 2006); (b) DNA binding interactions by the L1 loop in monomer D. The same interactions are also seen in monomer B but not in monomers A and C. (c) Arg248 of monomers B and D (not shown) adopts an extended conformation in the minor groove and makes a water-mediated hydrogen bond to N3 of the fifth adenine from the neighboring quarter site. (d) Arg248 of monomers A and C (not shown) flips out of the minor groove to interact with the DNA backbone. The electron density of Arg248 in (c) and (d) is calculated from simulated annealing omit map and contoured at 3 e/ų level.

Figure 5. The dimer-dimer interface

(a) Structural elements from monomer D (green) and monomer A (blue) forming the dimer-dimer interface are shown in a transparent surface model. The two patches of the interface (Patch I and II) are separated by a large solvent cavity; (b) The detailed interactions at Patch I viewed from above the dimer-dimer interface (Patch II is removed in this view for clarity). The van der Waals spheres of interface residues are shown as dotted spheres and are labeled by the color of their respective monomers (green: monomer D; blue: monomer A). The hydrogen bonds mediated by two ordered water molecules (red spheres) at the interface are also indicated; (c) The detailed interactions at Patch II viewed from the side the dimer-dimer interface (the same view as Figure 1a); (d) Sequence alignment of p53 from several representative species. Red boxes indicate residues at the dimer interface. Blue boxes indicate residues at the dimer-dimer interface on the 5′ side (monomers A and C). Green boxes indicate residues at the dimer-dimer interface on the 3′ side (monomers B and D).

Figure 6. Structural comparison with other p53:DNA complexes

(a) The tetramer is superimposed with the chemically trapped complex (gray, 3EXJ.pdb) using the Cα of monomer A as the reference (Malecka et al., 2009). The DNA of 3EXJ.pdb is omitted for clarity; (b) A detailed comparison using Cα backbone overlay to show the structural shift at the dimer-dimer interface. Upper panel: viewed from the side of the dimer-dimer interface; lower panel: viewed from the top of the dimer-dimer interface; (c, d) The structure of a p53 dimer bound to DNA (cyan, 2ATA.pdb) is superimposed on the A-B dimer of the tetramer using the Cα of monomer A as the reference (Kitayner et al., 2006). The rotational shift of the dimer partner (monomer B) can be observed from the side of the dimer-dimer interface (c) as well as above the interface (d).