Fig. 7. Sedimentation gradient of HPV18+ HeLa cells indicate that 18E6 is fully occupied at endogenous levels.
a SPR measurements of MPB-18E6 binding to biotin-E6AP at varying concentrations are shown. The black line represents the mean binding response of three independent experiments, while the red line represents the curve fit to a 1:1 interaction model. KD, dissociation constant; ka, association rate, kd, dissociation rate. b HeLa lysate was fractionated by 20–60% sucrose gradient at 367,600 g for 24 h at 4 °C, and fractions analyzed by SDS-PAGE and blotted for 18E6 (upper blot) or E6AP (lower blot). The quantification of 18E6 (red circles) or E6AP (black squares), with values representing the mean of two independent experiments, is shown above the blots. The peak fractions for fluorescent Myoglobin at 17 kDa, Ovalbumin at 44 kDa, BSA at 66 kDa, and Phosphorylase B at 97 kDa are also shown above. c Schematic diagram illustrates the 16E6 and E6AP-mediated degradation of p53. After 16E6 integration into the host genome, 16E6 is expressed and recruited by E6AP through its picomolar binding affinity. (i-ii) The AlphaFold2 E6AP model suggests the flexibility of the E6AP LXXLL motif, and is depicted as a transition state. (iii) After 16E6:E6AP complex formation, (iv) p53 is recruited, ubiquitinated (Ub, orange circles), and (v) degraded by the proteasome which promotes cell survival.