Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation

Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2363-8. doi: 10.1073/pnas.0436813100. Epub 2003 Feb 21.

Abstract

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Western
  • CDC2-CDC28 Kinases*
  • Calorimetry
  • Cell Cycle Proteins*
  • Crystallography, X-Ray
  • Cyclin D
  • Cyclin E / chemistry
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases / chemistry
  • Cyclins / chemistry
  • DNA-Binding Proteins*
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • Escherichia coli / metabolism
  • Glutathione Transferase / metabolism
  • Humans
  • Models, Molecular
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Retinoblastoma Protein / chemistry
  • Retinoblastoma Protein / metabolism*
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism

Substances

  • Cell Cycle Proteins
  • Cyclin D
  • Cyclin E
  • Cyclins
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • Recombinant Fusion Proteins
  • Retinoblastoma Protein
  • Transcription Factors
  • Glutathione Transferase
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases

Associated data

  • PDB/1O9K