Investigating the interaction of E6/E6AP heterodimer with p53-DBD by fluorescence resonance energy transfer

Human papilloma viruses (HPV) are small DNA viruses, among which the type 16 is the most prevalent and responsible for about 50% of human cervical carcinomas. The E6 oncoprotein of HPV-16 interacts with numerous host proteins by recognizing their LXXLL motifs. One of the HPV-16 E6 cellular binding targets is the E3 ubiquitin ligase E6AP. Once binding, the E6/E6AP heterodimer recruits and ubiquitinates p53, leading to subsequent degradation of p53. Importantly, neither E6 nor E6AP interacts with p53 alone. Therefore, blocking the interaction between E6 and E6AP could stabilize p53. Indeed, a LQELL peptide has been found capable to interact with E6 and suppress degradation of p53. Interaction of ligands with p53 may also reduce or inhibit binding of p53 with E6. While the crystal structure of E6/E6AP/p53 has been determined, efficient ligands targeting E6/E6AP/p53 are still missing. In this work, we studied the interactions between E6/E6AP fusion protein and p53 DNA binding domain using fluorescence resonance energy transfer (FRET). E6/E6AP fused to YFP and p53 fused to CFP were expressed from E. coli and purified using Ni affinity chromatography. We characterized the complex stability and obtained a dissociation constant consistent with previously determined by isothermal titration calorimetry. We further obtained the rate constant for dissociation of p53 from E6/E6AP using unlabeled p53 protein. Since the intensity of FRET signal is related to the amount of E6/E6AP/p53 complex, we further investigated the effect of p28, a p53 binding peptide, on the stability of E6/E6AP/p53 complex. Our preliminary results showed that p28 interferes with the binding of p53 to E6/E6AP. © FASEB.