Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopy.
Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour-suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down-regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N-terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein-protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti-cancer mechanism adopted by azurin.
Funari
Gloria
G
Biophysics and Nanoscience Centre, CNISM, Facoltà di Scienze, Università della Tuscia, Viterbo, Italy.
Domenici
Fabio
F
Nardinocchi
Lavinia
L
Puca
Rosa
R
D'Orazi
Gabriella
G
Bizzarri
Anna Rita
AR
Cannistraro
Salvatore
S
D001400:Azurin / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D001400:Azurin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2