CONICET | Buscador de Institutos y Recursos Humanos

Membrane-less organelles are formed by biomolecular condensation of macromolecules driven by the physicochemical process known as liquid-liquid phase separation (LLPS), where further transitions from liquid to solid states impact on a variety of physiological and pathological processes. The formation of ordered aggregates of the tumour-suppressor protein p53, suggests that LLPS plays a key role in its regulation and activities. p53 has been shown to inhibit replication of human papillomaviruses (HPV), and the HPV E2 proteins can induce p53-dependent apoptosis, both processes involving E2-p53 interactions whose molecular details are largely unknown. By using spectroscopic techniques combined with fluorescence microscopy, here we show that full-length p53 undergoes homotypic LLPS at neutral pH and low ionic strength. In addition, we show that p53 and the HPV16 E2 DNA binding domain (E2C) readily form heterotypic liquid-like droplets. Time-course experiments revealed that the homotypic droplets evolved to amyloid-like aggregates, whereas large spherical droplets containing both proteins remain up to 24 hours. When assessing the effect of specific target DNA duplexes, we found that p53 bound to its specific DNA fail to form droplets or condensates. On the other hand, the complex formed by p53 and its specific DNA recruited E2 to form large solid-like amorphous aggregates. Lastly, cellular transfection experiments showed that p53 and E2 co-localize in the nucleus in a granular chromatin-associated pattern, strongly suggestive of phase-separated condensates. This work demonstrates the capacity of p53 to follow different condensation routes as part of its role in cellular processes and specially highlights the formation of functional condensates and their intertwined connection with gene function control in a cancer-associated virus.