title: Azurin modulates the association of Mdm2 with p53: SPR evidence from interaction of the full-length proteins.
abstract: The tumour suppressor p53 plays a crucial role in cell stress response and its anticancer activity is mainly down-regulated by the oncoprotein Mdm2 that, upon binding to p53, blocks its transcriptional activity and promotes its ubiquitin-dependent degradation. Targeting Mdm2-p53 interaction is believed to be the most direct of all p53-activating strategies to treat tumours in which p53 has retained its wild-type function. The bacterial protein Azurin has been shown to bind p53, inhibiting cancer cell proliferation likely through a post-translational increasing of the p53 level. This apparent antagonist action with respect to the Mdm2-p53 functional interaction suggests that binding of Azurin to p53 might interfere with the Mdm2-p53 association and, thus, preventing p53 from degradation. Toward this end, a detailed kinetic characterization of the binding interaction of these three proteins has been performed by surface plasmon resonance. The occurrence of specific binary interactions of both Azurin and Mdm2 with p53, as investigated more appropriately in their full-length conformation, is ascertained and the corresponding association and dissociation rate constants are measured. Interestingly enough, the three proteins are likely engaged in a ternary interaction, whose kinetics points out that binding of Azurin to p53 causes a significant decrease of the Mdm2-p53 association rate constant and binding affinity, without hindering the accessibility of Mdm2 to the binding pocket of p53. The Azurin-induced p53 conformational change, as demonstrated by circular dichroism, suggests that the protein may affect Mdm2-p53 association through an allosteric mechanism, which could give an useful insight into designing new anticancer drugs.
authors.affiliation: Biophysics and Nanoscience Centre, CNISM, Facoltà di Scienze, Università della Tuscia, Viterbo, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Domenici
authors.forename: Marco
authors.initials: M
authors.lastname: Frasconi
authors.forename: Franco
authors.initials: F
authors.lastname: Mazzei
authors.forename: Gabriella
authors.initials: G
authors.lastname: D'Orazi
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Bizzarri
authors.forename: Salvatore
authors.initials: S
authors.lastname: Cannistraro
mesh_terms: D001400:Azurin / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D016159:Tumor Suppressor Protein p53; D001400:Azurin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2