Macromolecular Modeling Blog ™

S. Gordo et al. implement a new strategy for preventative anti-cancer therapeutics. R337H is the most frequent inherited mutation in p53, “The guardian of the genome”, and is associated with wide spectrum of cancer forms. The mutation is believed to destabilize the native tetramer form of p53. In this study the researchers designed a small molecule that reconstitutes tetramerization of such a mutant. By Nir London.

Susana Gordo, Vera Martos, Eva Santos, Margarita Menendez, Carles Bo, Ernest Giralt and Javier de Mendoza.

It has been shown that mutations compromising the ability to form the tetrameric functional structure in the tumor suppressor protein p53 may end in cancer development. For instance, the most frequent inherited mutation, p53-R337H, is associated to adrenocortical carcinoma, as well as to a wide spectrum of other cancer forms. Mutated p53-R337H cannot efficiently assemble the active tetramer and, consequently, leads to an organism prone to tumor setup. The activity of the protein strongly depends on its tetrameric integrity. Hence, molecules able to stabilize the tetrameric structure of mutated proteins with compromised tetramerization abilities could be valuable therapeutical tools.

The structure of p53TD (tetramerization domain) was elucidated both by x-ray crystallography and NMR. Two identical chains of 31 residues folded into single alpha-helix-beta-hairpin secondary structures associate to form intertwined dimers, which in turn stack into tetramers. Hydrogen bonding, hydrophobic interactions, and salt bridges stabilize the dimer-to-dimer association. One of these interactions, namely the ion-paired bridge Arg-337-Asp-352 between two monomers is lost in mutant p53-R337H, and the tetramer is destabilized, because the histidine is not fully protonated at physiological conditions.

Taking advantage of the spatial arrangement of E336 and E339 residues in strands of two different monomers, whose free carboxylate side chains form an almost perfect square just above the hydrophobic pocket located within the strands, the researchers hypothesized that a hydrophobic, conically shaped molecule endowed with four cationic residues at suitable distances to the above-mentioned anionic residues would efficiently complement the shape of the protein tetramer, thus stabilizing the whole ensamble.

The authors designed exactly such a molecule (a conical calix[4]arene platform with four guanidiniomethyl residues) named “Ligand 1″ and validated its stabilization of the the tetramer in various ways.

Molecular Dynamics: Protein and protein-ligand stabilities were assessed by analyzing the trajectories obtained in 10-ns molecular dynamics simulations at 300 and 400 K, aimed at simulating thermal denaturing conditions. In the absence of ligand, the structure of wild-type p53TD was highly stable at either 300 or 400 K over the whole dynamics. However, for mutated p53TD-R337H striking differences were observed. At 300 K, the rmsd grows gradually along the trajectory, never recovering its initial value. At thermal denaturing conditions (400 K), the differences between wild and mutated proteins were evident from the very early steps of the trajectory.

Ligand 1, docked on the protein, remained stable at the same site it was initially positioned, interacting with the protein through hydrogen bonds and hydrophobic interactions. Most remarkably, whereas ligand binding did not significantly influence the rmsd of wild-type p53TD, it strongly stabilized mutant p53-R337H, as the decrease in the rmsd value and the attenuation of the fluctuations clearly showed.

Stability Effects: Experimentally, the thermal effects caused by ligand 1 on the stability of both wild-type and mutated proteins were evaluated by circular dichroism and differential scanning calorimetry (DSC). The presence of 400 microM of calixarene 1 hardly shifted a couple of degrees the DSC unfolding curve of the highly stable wild-type p53TD (from 85.5°C to 86.9°C), which indicated that, at the melting temperature, the calixarene ligand displayed rather low affinity for the stable protein.

Conversely, ligand 1 induced an outstanding thermal stabilization in mutant p53TD-R337H, which beyond a shift toward higher melting temperatures (from 62°C to 82°C) resulted also in a substantial increase in the unfolding enthalpy. Thermal changes detected on the mutant protein not only indicated a tight protein-ligand interaction, but also suggested that the tetramer was the actual stabilized structure, in full agreement with the molecular dynamic simulations described above.

Structural Characterization: Structural aspects were examined by NMR, on both the ligand and the protein. These experiments not only determined that p53TD and ligand 1 interacted specifically, but also mapped the interacting molecules. Results completely agreed with the design rationale for ligand 1, with the hydrophobic lower rim of the calixarene ‘‘inserted” like a cork into the protein cleft, whereas the upper rim remained outside. A thorough titration of 15N-p53TD with ligand 1, followed by 1H-15N-heteronuclear sequential quantum correlation (HSQC) proved which parts of the protein structure were directly involved in the interaction. The protein binding site was in perfect agreement with the design model.

However when a similar titration was carried out for mutant p53TD-R337H, a totally different evolution for the HSQC spectra occurred, indicating that interaction of ligand 1 with p53TD-R337H took place through a different and more complex mechanism. Likely, the histidine-mutated residue, as a part of the hydrophobic pocket, might cause significant distortions affecting the binding of the ligand with respect to the wild-type protein mode.

These complex behaviors could be interpreted considering two sequential events, each of them probably corresponding to the binding of a molecule of calixarene ligand to the tetramer. The fact that any clear intermediate was not detected suggested that the second binding should be of larger affinity than the first one, that is, the two molecules of calixarene bind sequentially in a cooperative manner. Indeed, assuming this mechanism, the dissociation constants for both binding events were estimated to be KD1: 130microM and KD2: 65microM.

The authors conclude that these results encourage strategies in drug discovery, based on the use of small molecules acting as templates for the structural recovery of the supramolecular integrity of damaged protein assemblies.

By Nir London.

S. Gordo, V. Martos, E. Santos, M. Menendez, C. Bo, E. Giralt, J. de Mendoza (2008). Stability and structural recovery of the tetramerization domain of p53-R337H mutant induced by a designed templating ligand Proceedings of the National Academy of Sciences, 105 (43), 16426-16431 DOI: 10.1073/pnas.0805658105

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