A CONSERVED, INTRINSICALLY DISORDERED AND FLEXIBLE ?LINKER" ALLOWS COOPERATIVE HIGH AFFINITY INTERACTION OF THE VIRAL E1A PROTEIN WITH ITS TARGET RETINOBLASTOMA

GLAVINA, J; CHEMES, L.B.; SANCHEZ, IE; GONZALEZ FOUTEL N; DE PRAT-GAY G

SANTIAGO DE CHILE

Simposio; SIMPOSIO Protein Biophysics at the end of the world; 2016

Pathogenic viruses like Adenovirus, HPV and SV40 harbor proteins able to interact with the retinoblastoma tumor suppressor (Rb), central controller of eukaryotic cell cycle. Such interactions target sites used by Rb to bind its endogenous targets: the E2F transcription factor-binding (E2F-mimic) and LxCxE sites. These viruses use linear motifs present in intrinsically disordered regions, to enable effective interference with the cellular network. The Adenovirus E1A viral oncoprotein (AdE1A) binds Rb through two linear motifs required to displace E2F. These binding motifs are located each in the ?CR1? and "CR2" E1A domains respectively, and are separated by a 70-residue "linker". Given the limited structural and mechanistic knowledge regarding these interactions, we set out to characterize AdE1A (CR1-CR2) and its interaction with Rb.For this purpose, we expressed the AdE1A (36-146) recombinant fragment containing the CR1 and CR2 domains, with 8 mg/liter final yield and ≥95% purity. Static light scattering and circular dichroism (CD) studies revealed that AdE1A (36-146) is an intrinsically disordered monomer with extended (Rh/MW ~ 4) hydrodynamic radius. Interaction assays show that E1A binds to the RbAB central domain with 1: 1 stoichiometry, with complex formation accompanied by a strong compaction of AdE1A. In-solution spectroscopic measurements revealed that the affinity of the [AdE1A: Rb] complex containing both binding sites (KD = 20 pM) is ~ 7000-fold enhanced compared to the individual sites (KD = 140nM), with a lifetime of over 2hr. A quantitative analysis based on polymer theory indicates that the cooperative effect can be explained by predictions of the effect of a flexible linker joining both sites. The length and intrinsically disordered nature of the "linker" is conserved across E1A strains within a range yielding optimum binding affinity, suggesting evolutionary conservation of linker properties that allows effective displacement of E2F.