Structure- and modeling-based identification of the adenovirus E4orf4 binding site in the protein phosphatase 2A B55? subunit.

ABSTRACT: BACKGROUND: The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions. RESULTS: The E4orf4 binding site in PP2A was mapped to the ?1,?2 helices of the B55? subunit. CONCLUSION: The E4orf4 binding site in PP2A-B55? lies above the substrate binding site and does not overlap it. SIGNIFICANCE: A novel functional significance was assigned to the ?1,?2 helices of the PP2A-B55? subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55? subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55? has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55? and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55?. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the ?1 and ?2 helices described in the B55? structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55? mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the ?1,?2 helices of B55?, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.

SUBMITTER: Horowitz B 

PROVIDER: S-EPMC3650409 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature