Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma tumor suppressor

Abstract

Repression of E2F transcription activity by the retinoblastoma (Rb) tumor suppressor through its interaction with the transactivation domain of the E2F transcription factor is one of the central features of G1/S arrest in the mammalian cell cycle. Deregulation of the Rb-E2F interaction results in hyperproliferation, lack of differentiation, and apoptosis, and can lead to cancer. The 2.2-A crystal structure of the Rb pocket complexed with an 18-residue transactivation-domain peptide of E2F-2 reveals that the boomerang-shaped peptide binds to the highly conserved interface between the A-box and the B-box of the Rb pocket in a bipartite manner. The N-terminal segment of the E2F-2 peptide in an extended beta-strand-like structure interacts with helices from the conserved groove at the A-B interface, whereas the C-terminal segment, which contains one 3(10) helix, binds to a groove mainly formed by A-box helices. The flexibility in the middle of the E2F-2 peptide is essential for the tight association of E2F to the Rb pocket. The binding of Rb to the E2F-2 peptide conceals several conserved residues that are crucial for transcription activation of E2F. We provide the structural basis for the Rb-mediated repression of E2F transcription activity without the requirement of histone-modifying enzymes.

Figure 1

(a) Electron density map for the bound E2F-2 peptide. The difference map was calculated using phases derived from the model after omitting the entire E2F-2 peptide and subjecting the model to simulated annealing refinement from 2000 K using CNS to remove model bias. The map was calculated at 2.2-Å resolution and contoured at 2.5 ς. (b) Overall structure of the Rb pocket in complex with the E2F-2 transactivation-domain peptide. The schematic representation of the right panel is related to the one on the left by a 90° rotation around the horizontal axis. (Blue) A-box in Rb; (red) B-box in Rb; (yellow) E2F-2. The secondary structure in the binding region in the Rb pocket is labeled.

Figure 2

Sequence of the E2F family showing secondary structure elements. Conserved residues are highlighted in yellow. The bar graph shows the buried surface area per E2F-2 peptide residue upon complex formation.

Figure 3

Surface representation of the Rb pocket colored according to the degree of conservation in the Rb orthologs of five species and p107 and p130 family members (Lee et al. 1998). The A–B interface and the A-box where E2F binds are shown. The conserved groove in the B-box where the LxCxE motif binds is in the opposite face (marked with an arrow and an asterisk shown in blue) and cannot be seen in this view.

Figure 4

Schematic representation of interactions between the Rb pocket and the E2F-2 transactivation-domain peptide. (a) A closeup view of the interactions between the N-segment of the E2F-2 peptide and helices α8 and α9 from the A-box and α11 from the B-box in the A–B interface of the Rb pocket. (Yellow) E2F-2 peptide; (blue) A-box; (red) B-box; (yellow) interacting side chains in E2f-2; (green) interacting side chains in Rb; (red) O atoms; (blue) N atoms. In addition to the van der Waals contacts at the interface, there are eight intermolecular H-bonds: Lys 548 NZ to Asp 410 OD2, His 555 NE2 to Asp 411 OD2, Glu 554 OE2 to Tyr 412 OH, Lys 653 NZ to Tyr 412 OH, Lys 653 NZ to Leu 413 carbonyl, Glu 533 OE1 to Gly 414 amide, Lys 652 NZ to Gly 414 carbonyl, and Glu 533 OE2 to Leu 415 amide. (b) A closeup view of the interactions between the C-segment of E2F-2 and helices α4, α5, α6, α8, and α11 of the Rb pocket. The conserved residues, Ile 422, Leu 425, and Phe 426, of E2F-2 form multiple van der Waals contacts with residues from the Rb pocket. In the Rb pocket, the interacting residues are Met 460 on helix α4; Leu 476 on helix α5; Ile 481 and Phe 482 on helix α6; Tyr 529, Lys 530, Val 531, and Ser 534 on helix α8; and Ser 646 and Leu 649 on helix α11. The color-coding scheme is as in a.

Figure 5

ITC titration curves for E2F-2 peptide binding to the Rb pocket. The interactions between the Rb pocket and intact E2F-2 peptide (a); a Glu420Ala mutant (b); a Gly421Ala mutant (c); and intact E2F-5 peptide (d) are measured.

Figure 6

Surface plasmon resonance experiments. Sensograms of E2F-1 binding to wild-type and mutant Rb proteins that were used to calculate the kinetic binding constants are shown. The sensogram data at 100 nM for each Rb protein are superimposed and displayed. (Magenta) wild-type Rb pocket; (yellow) Arg467Ala mutant; (red) Phe482Ala mutant; (blue) Glu554Ala mutant; (green) Lys653Ala mutant.

Figure 7

Conserved E2F residues that make contact to Rb are involved in transcription activation. (a) Five conserved residues—Tyr 412, Trp 414, Ile 422, Leu 425, and Phe 426—fit tightly into the A–B interface of the Rb pocket. The Rb surface is represented as a wire mesh, and E2F-2 residues are in space-filling representation. (b) The E2F-dependent transactivation potential for wild-type and mutant E2F-1. Each of wild-type and mutant E2F-1 was transfected into SAOS-2 cells and its ability to activate the expression of luciferase gene was assayed. Double, triple, and multiple E2F mutants were used for the activity measurements (see text).

Figure 7

Conserved E2F residues that make contact to Rb are involved in transcription activation. (a) Five conserved residues—Tyr 412, Trp 414, Ile 422, Leu 425, and Phe 426—fit tightly into the A–B interface of the Rb pocket. The Rb surface is represented as a wire mesh, and E2F-2 residues are in space-filling representation. (b) The E2F-dependent transactivation potential for wild-type and mutant E2F-1. Each of wild-type and mutant E2F-1 was transfected into SAOS-2 cells and its ability to activate the expression of luciferase gene was assayed. Double, triple, and multiple E2F mutants were used for the activity measurements (see text).

Figure 8

Contacts made by the E2F-2 peptide with the conserved B-box in the Rb pocket where the viral protein LxCxE motif binds. The E2F-2 peptide (yellow) is superimposed with a nine-residue HPV E7 peptide containing the LxCxE motif (blue). (Red) Helices in B-box where the conserved Leu, Cys, and Glu in the E7 peptide bind; (magenta) helices from the other B-box in the asymmetric unit.