p53 Thermodynamic Stability

Thr-123 is located at the end of the flexible L1 loop that makes contacts with the DNA major groove via Lys-120. The Thr-123 side chain is solvent exposed. Its hydroxyl group makes no direct but water-mediated intramolecular contacts. Apart from the truncated side chain (threonine to alanine), no significant T123A-induced structural changes are observed in the DNA-free crystal structure of the heptamutant T123A/M133L/H168R/
V203A/N239Y/R249S/N268D (PDB 2BIQ) when compared with the structure of the corresponding hexamutant without T123A (PDB 2BIP).

Met-133 is located on β-strand S2’ of the loop-sheet-helix motif. Via its side-chain it contributes to the central hydrophobic core of the protein. The M133L mutation induces only minor structural changes in the immediate environment of the mutation and leaves the overall structure of the hydrophobic core intact (PDB 1UOL).

Val-143 is located on β-strand S3. Its side chain is part of the central hydrophobic core of the β-sandwich. The V143A mutation creates an internal cavity in the hydrophobic core of the β-sandwich without collapse of the surrounding structure. The mutant is highly destabilized as a result of lost hydrophobic interactions due to the two truncated methyl groups (PDB 2J1W).

The highly destabilizing H168R mutation induces substantial structural distortion around the mutation site in the L2 loop at the periphery of the DNA-binding surface. Several residues including the mutation site are disordered in the crystal structure; i.e. no defined conformation is observed for residues 166-170 and the side chain of Glu-171 (PDB 2BIN).

Val-203 is located in the turn connecting the β-strands S5 and S6. Mutation to alanine has no effect on the overall structure of the protein apart from conformational changes of adjacent side chains (PDB 1UOL).

The Y220C mutation is located at the far end of the β-sandwich, at the start of the loop connecting β-strands S7 and S8. This highly destabilizing mutation creates an extended surface crevice but retains the structural features of the wild type in functionally important surface regions (PDB 2J1X).

Asn-239 is located at the beginning of the L3 loop in the immediate vicinity of the zinc-binding site. Its side chain is solvent exposed and makes water-mediated contacts with the DNA backbone upon binding of specific p53 response elements (e.g. PDB 2AC0). The N239Y mutation stabilizes the protein and rigidifies the local structure without perturbing it. It creates novel hydrophobic packing interactions between the zinc-binding region and Leu-137 at the edge of the loop-sheet-helix motif (S2’/S3 loop) (PDB 1UOL).

Gly-245 is located in the L3 loop, close to the zinc-binding site. It adopts a main-chain conformation that is not favored in non-glycine residues. Upon G245S mutation, the serine side chain is accommodated by displacing a structural water molecule that is conserved in the wild type and other mutant structures. The mutation is accompanied by small but significant changes in the backbone conformation of the L3 loop. The structural changes affect residues that form part of the self-complementary core domain-core domain dimerization interface in the p53-DNA complex. Presumably G245S impairs DNA binding by reducing binding cooperativity (PDB 2J1Y).

The guanidinium group of Arg-249 makes crucial interactions that stabilize the hairpin conformation of the L3 loop, which docks the core domain to the minor groove of DNA-response elements via Arg-248. It forms a salt bridge with Glu-171 and hydrogen bonds with the backbone oxygens of Gly-245 and Met-246. Upon mutation to serine, these interactions are lost. The L3 loop becomes highly flexible and undergoes a large conformational change, favoring a non-native conformation. Both DNA contacts (Arg-248) and residues that form the self-complementary core domain-core domain interface upon DNA binding (e.g. Met-243 and Gly-244) are affected. Most notable is a switch of methionines 243 and 246. Met-243 is located on the surface of the core domain in the wild type. In the R249S mutant, this region adopts a helical conformation, and the side chain of Met-243 is buried in the interior of the protein by displacing the side chain of Met-246 from this buried position (PDB 2BIO).

Phe-270 is located on β-strand S10. Its side chain is an integral part of the central hydrophobic core of the β-sandwich. The F270L mutation creates an internal cavity in this hydrophobic core without collapse of the surrounding structure. The mutant is highly destabilized as a result of the truncated hydrocarbon moiety (PDB 2J1Z).

R273C removes the essential DNA-contact residue Arg-273 that contacts the phosphate backbones at the center of a DNA half-site. The conformation of neighboring side chains (e.g. Phe-134, Ser-240 or Asp-281) has not significantly changed in the mutant crystal structure, i.e. the overall architecture of the DNA-binding surface is preserved (PDB 2J20).

R273H removes the essential DNA-contact residue Arg-273 that contacts the phosphate backbone at the center of a DNA half-site. The conformation of neighbouring side chains (e.g. Phe-134, Ser-240 or Asp-281) has not significantly changed in the mutant crystal structure, i.e. the overall architecture of the DNA-binding surface is preserved (PDB 2BIM).

In the wild-type protein, Arg-282 stabilizes the loop-sheet-helix motif via a network of hydrogen bonds and hydrophobic interactions (e.g. hydrogen bonds with the hydroxyl group of Thr-125 and the backbone oxygen of Tyr-126, and a salt bridge with Glu-286). These interactions are lost in the R282W mutant, which is substantially destabilized as a result. The tryptophan side chain causes steric hindrance and perturbs the L1 loop, which includes the DNA-contact residue Lys-120. The overall architecture of the remainder of the DNA-binding region is conserved, consistent with the observation that the R282W mutant binds to several promoters at sub-physiological temperature at which the mutant is folded (PDB 2J21).

The H168R/R249S double mutation largely reverses the structural perturbations caused by the single point mutations H168R and R249S. The guanidinium group of Arg-168 in the double mutant mimics the structural role of Arg-249 in the wild type that is key to stabilizing the hairpin conformation of the L3 loop required for sequence-specific DNA binding. As a result, the wild type conformation of the L2/L3 loop region is largely restored, and wild-type like DNA-binding properties are observed in the folded state (PDB 2BIP and 2BIQ).