Binding kinetics of mutant p53R175H with wild type p53 and p63: A Surface Plasmon Resonance and Atomic Force Spectroscopy study

摘要

Abstract The oncogenic mutant p53R175H, one of the most frequently occurring in human cancers and usually associated with poor prognosis and chemo resistance, can exert a dominant negative effect over p53 family members, namely wild type p53, p63 and p73, inhibiting their oncosuppressive function. Novel anticancer strategies based on drugs able to prevent the formation of complexes between p53R175H and the p53 family members call for a deeper knowledge on the molecular mechanisms of their interaction. To this aim, p53R175H/p63 and p53R175H/p53 complexes were investigated in vitro by using Surface Plasmon Resonance and Atomic Force Spectroscopy, two emerging and complementary techniques able to provide interaction kinetic information, in near physiological conditions and without any labelling. Both approaches show that p53R175H forms a very specific and highly stable bimolecular complex with both p63 and p53; with these interactions being characterized by a very high affinity with equilibrium dissociation constant, K D , of about 10 ? 9 M. These kinetics results, discussed also in connection with those previously reported for the interaction of p53R175H with p73, could inspire the design of suitable anticancer drugs able to antagonize the interaction of p53R175H with the p53 family members, by restoring then their anti-tumour function. Graphical abstract Display Omitted Highlights ? SPR and AFS monitored the high affinity complex of p53R175H and p53wt or p63. ? Mutant p53R175H interacts with all p53 family (p53, p63 and p73) with high affinity. ? Measured kinetics are insightful to drug design inhibiting these complexes.