title: Structure- and modeling-based identification of the adenovirus E4orf4 binding site in the protein phosphatase 2A B55α 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 α1,α2 helices of the B55α subunit.
  
  
  CONCLUSIONS
  The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it.
  
  
  CONCLUSIONS
  A novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α 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 B55α 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-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α 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 B55α 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 α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.
authors.affiliation: Department of Molecular Microbiology, Faculty of Medicine, Technion-Israel Institute of Technology, Bat Galim, Haifa 31096, Israel.
authors.forename: Ben
authors.initials: B
authors.lastname: Horowitz
authors.forename: Rakefet
authors.initials: R
authors.lastname: Sharf
authors.forename: Meirav
authors.initials: M
authors.lastname: Avital-Shacham
authors.forename: Antonina
authors.initials: A
authors.lastname: Pechkovsky
authors.forename: Tamar
authors.initials: T
authors.lastname: Kleinberger
mesh_terms: D017504:Adenovirus E4 Proteins / Q000737:chemistry*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D054648:Protein Phosphatase 2 / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D051381:Rats; D040681:Structural Homology, Protein
chemical_list: D017504:Adenovirus E4 Proteins; C582184:Ppp2r2a protein, rat; D054648:Protein Phosphatase 2
keywords: Adenovirus; Bioinformatics; PP2A; Protein-Protein Interactions; Signal Transduction