This study presents a set of experiments where different approaches in structural mass spectrometry were applied in order to obtain insight into how (i) the E6 oncoprotein of cancer-associated HPVs stimulates the E3 ligase activity of E6AP, and (ii) E6AP, E6, and the tumor suppressor p53 are structurally and functionally arranged within the E6AP–E6–p53 enzyme–substrate complex. Here, UBE3A is linked to cancer.