de Prat Gay, G. | Search bioRxiv

bioRxiv2022

The guardian of the genome meets a viral master gene regulator at a biomolecular condensate

As guardian of the genome, p53 exerts its tumor suppressor activity by modulating the expression of several hundreds of genes and by interacting with a large number of proteins. However, p53 can also repress viral replication and it is targeted by a variety of viral proteins to allow viral replication to proceed. p53 can repress human papillomavirus replication by binding to the viral E2 master gene regulator. Here we show how full-length p53 can spontaneously form phase separated liquid-like droplets that evolve to amyloid-like aggregates in a time-dependent manner, highlighting the fact that homotypic condensation is on the path to aggregation as observed in several protein aggregopathies. The DNA binding domain of HPV E2 (E2C) triggers heterotypic liquid-liquid phase separation with p53 with a precise 1 p53 : 2 E2C stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Moreover, E2C is able to slowly reshape time-evolved p53 aggregates into regular heterotypic liquid droplets. Using in situ sub-cellular fractionation, we show that E2 and wild-type p53 co-localize to the nucleus with a grainy pattern, and E2 can re-localize p53 into chromatin associated foci, a function independent of the DNA binding capacity of p53. A small DNA duplex containing the specific binding site for p53 deforms and dissolves both homotypic and heterotypic condensates at a 1 p53 : 1 DNA stoichiometry, whereas a [~]1000 base pair DNA fragment instead reshaped the condensates into distinct amorphous condensates containing p53, E2C and DNA, reminiscent of what we observe bound to chromatin. We conclude that p53 is a scaffold for liquid-liquid phase separation in line with its structural and functional features, in particular as a hub that binds multiple cellular protein partners as well as nucleic acids. Moreover, the capacity of E2C to rescue p53 from the amyloid aggregation route impacts on p53-rescuing drugs cancers where p53 mutation leads to loss of function.

biophysics↗

bioRxiv2021

Conformational buffering underlies functional selection in intrinsically disordered protein regions

Many disordered proteins conserve essential functions in the face of extensive sequence variation. This makes it challenging to identify the forces responsible for functional selection. Viruses are robust model systems to investigate functional selection and they take advantage of protein disorder to acquire novel traits. Here, we combine structural and computational biophysics with evolutionary analysis to determine the molecular basis for functional selection in the intrinsically disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, triggering early S-phase entry and disrupting normal cellular proliferation. We show that the ability to outcompete host factors depends on the picomolar binding affinity of E1A for Rb, which is driven by two binding motifs tethered by a hypervariable disordered linker. Binding affinity is determined by the spatial dimensions of the linker, which constrain the relative position of the two binding motifs. Despite substantial sequence variation across evolution, the linker dimensions are finely optimized through compensatory changes in amino acid sequence and sequence length, leading to conserved linker dimensions and maximal affinity. We refer to the mechanism that conserves spatial dimensions despite large-scale variations in sequence as conformational buffering. Conformational buffering explains how variable disordered proteins encode functions and could be a general mechanism for functional selection within disordered protein regions.

biophysics↗