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. 2017 May 9;114(19):4942-4947.
doi: 10.1073/pnas.1619170114. Epub 2017 Apr 24.

Conservation and Divergence of C-terminal Domain Structure in the Retinoblastoma Protein Family

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Free PMC article

Conservation and Divergence of C-terminal Domain Structure in the Retinoblastoma Protein Family

Tyler J Liban et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Keywords: E2F; cell cycle; evolution; protein–protein interactions; tumor suppressor protein.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Interactions between pocket protein C-terminal domains and the E2F coiled-coil and marked-box domains. (A) Domain architecture of pocket proteins, E2F, and DP. The E2F transactivation domain binds the pocket domain, and the E2F–DP CM domain binds the CTD. (B) Sequence alignment of the human E2F CM domains and pocket protein CTDs. The secondary structure elements present in our crystal structures are indicated along with residues in E2F5 (*) that contact the p107 CTD. Cdk phosphorylation sites are indicated with blue dashed outlines. (C) Calorimetry measurements of RbC and p107C binding to E2F–DP CM domains. p107994–1031 binding was measured here, whereas the other values were previously reported (19). The asterisk indicates that the measurement was made with E2F4–DP2CM.
Fig. S1.
Fig. S1.
Coprecipitation assay identifying a minimal p107 CTD fragment that binds E2F4–DP1CM. One hundred micrograms of the indicated GST–p107 fragment and 100 μg of purified E2F4–DP1CM were incubated on ice for 1 h in a reaction volume of 200 μL containing 100 mM NaCl, 25 mM Tris, and 5 mM DTT (pH 8.0). Proteins were affinity-precipitated with glutathione Sepharose resin, washed, and eluted in the binding buffer plus 10 mM glutathione. For each binding reaction, the eluate containing bound proteins (B) was loaded onto an SDS polyacrylamide gel along with a sample of the unbound (U) reaction.
Fig. 2.
Fig. 2.
Crystal structures of the E2F4–DP1 and E2F5–DP1 CM domains. (A) Overall structures show similar topologies with the E2F and DP chains forming an extensive interface. (B) Overlay of the CM domain structures determined for E2F1–DP1 [gray; Protein Data Bank (PDB) ID code 2AZE], E2F4–DP1 (pink), and E2F5-DP1 (red and purple correspond to the two different molecules in the asymmetric unit). Structures were overlaid by alignment of β-sandwich domain Cα atoms (Fig. S2) so that the different positions of the coiled-coil domains reflect their different orientations relative to the β-sandwich domain. Only the β-sandwich domain of E2F1–DP1 is shown.
Fig. S2.
Fig. S2.
Conservation of the E2F–DP CM structure. Comparison of CM β-sandwich domains reveals similar structures. (A) Alignment of E2F4–DP1CM and E2F1–DP1CM structures. The root-mean-square deviation (rmsd) of the Cα position is 0.77 Å. (B) Alignment of E2F5–DP1CM and E2F1–DP1CM structures. Rmsd is 1.24 Å. (C) Alignment of E2F5–DP1CM and E2F4–DP1CM structures. Rmsd is 1.35 Å. (D) Alignment of two different E2F5–DP1CM heterodimers in the asymmetric unit of E2F5–DP1–p107C crystals. Rmsd is 1.14 Å. (E) E2F4–DP1CM is shown with residues that are conserved among E2F paralogs in yellow. For sequence alignment, see Fig. 1B. Most of the 20 residues that are identical among family members are critical for the structural core of the domain. A group of highly conserved residues forms the last E2F strand (β5) and the preceding loop. Their side chains form a surface on the edge of the sandwich opposite the edge that binds pocket proteins (Fig. 2A). The conservation here suggests that the exposed hydrophobic cleft along this sandwich edge is a potential protein interaction surface common to all E2F family members.
Fig. 3.
Fig. 3.
Comparison of p107C–E2F5 and RbC–E2F1 binding interfaces. (A) p107C binds the E2F5–DP1 β-sandwich domain using a strand-loop-helix motif and forms a hydrophobic interface with residues from both E2F and DP. (B) Overlay of RbC (taken from the RbC–E2F1 structure) with p107C models how RbC would bind E2F5. (C) Overlay of E2F1 and E2F5 models how p107C would bind E2F1. (D) Affinity measurements for p107C994–1031 binding to the indicated E2F–DPCM domains. The E2F4–DP1CM AVDS mutant has the E2F4 VPIP sequence (residues 167 to 170) mutated to AVDS, whereas the E2F1–DP1CM mutant has the E2F1 AVDS sequence (residues 275 to 278) mutated to VPIP. (E) Affinity measurements of an RbC–p107C hybrid protein containing residues 771 to 794 of Rb (RbCnter) fused to residues 975 to 1031 of p107.
Fig. S3.
Fig. S3.
Cancer-associated mutations in p107C. We used the Cancer Genome Atlas (https://cancergenome.nih.gov) to identify cancer-associated mutations in p107 and p130 that are localized to the CTD, and we mapped these mutations onto the crystal structure. Several missense mutations have been found at I1021 in p107 (I1092 in p130), which is found on the p107C helix and is buried into the E2F5–DP1CM sandwich core. We found that an I1021A mutation weakens the affinity of p107C for E2F4–DP1CM 30-fold, whereas an I1021M mutation found in colorectal cancer has a relatively modest 2-fold effect. We used E2F4 in our binding measurements because E2F4 is more abundant in cells and expresses well as a recombinant protein. E2F4 and E2F5 are highly conserved in the β3-strand that binds p107C (Fig. 1B), and both bind wild-type p107C with similar affinity (Fig. 3D). Most of the other mutations map to the surface of the p107C strand and helix that does not form the interface with E2F5–DP1CM. We did find that two mutations found in uterine cancer, K1012E in p107 and R1093C in p130 (R1022C in p107), and one mutation found in both colorectal and prostate cancer, R1093H in p130 (R1022H in p107), have two- to threefold effects on the complex affinity. We conclude that these mutations only slightly impair the ability of p107/p130 to bind E2F. However, an interesting alternative possibility, given the localization of these residues to the exposed face of the p107C helix, is that these mutations interfere with other protein interactions. Notably, this region of p107/p130 has been implicated in regulation of protein stability, and mutation of K774 in Drosophila (K1012 in human p107) leads to a developmental defect from loss of regulation of protein levels (23). In addition to E2F binding, the C-terminal helix may mediate protein interactions that control p107/p130 degradation.
Fig. S4.
Fig. S4.
RbC associates similarly with E2F1–DPCM and E2F3–DPCM, but the Rb pocket domain binds the transactivation domains (TDs) differently. It has been proposed that the RbC association is specific to E2F1 (11, 25, 26). One observation supporting this specificity is that a mutation in the pocket domain (R467E/K548E) at the transactivation-binding site inhibits Rb complex formation in cell extracts with E2F3 but not E2F1 (25, 26). We measured the affinity of RbC771–928 for E2F3–DP1CM and found that it is similar to the RbC affinity for E2F1–DP1CM and E2F4–DP1CM that we previously reported (Fig. 1C) (19). We considered that the Rb–E2F1 specificity observed in coimmunoprecipitation experiments in cell extracts is due to differences in other interactions outside the C terminus. For example, the E2F1 and E2F2 TDs bind the Rb pocket domain with significantly higher affinity than other E2Fs (27). We measured the affinity of the R467E/K548E Rb mutant for both the E2F1 and E2F3 TDs by ITC. Measurements of the Rb pocket domain (residues 380 to 787) mutant for the E2F1 TD (residues 409 to 426) and E2F3 (residues 432 to 449) are reported here. Wild-type measurements were previously reported (27). We find that whereas the mutations abolish E2F3 binding completely, we still observe some weak affinity for E2F1. We suggest that such differences in transactivation domain binding and not E2F–DPCM binding explain the preference of Rb for E2F1 in cells when probed using Rb constructs that contain E2F-binding mutations in the pocket domain (25, 26). We believe that the correct interpretation of the specific affinity previously observed between the pocket domain (R467E/K548E) mutant and E2F1 in cells is that the mutant still has some residual affinity for the E2F1 TD and not that only E2F1 interacts with RbC.
Fig. 4.
Fig. 4.
Phosphorylation of Cdk sites in p107C directly inhibits E2F binding. (A) ITC measurements of p107C peptides phosphorylated at the indicated sites. (B) Structure of the p107C–E2F5–DP1 interface.
Fig. 5.
Fig. 5.
Phylogenetic distribution of pocket protein and E2F subfamilies. Detected pocket proteins (Dataset S1) and E2Fs (Dataset S2) from each genome are classified into subfamilies based on phylogenetic analysis (SI Materials and Methods). The resulting distribution of homologs was condensed and ordered according to the current consensus on phylogenetic relationships between major animal lineages (36), including alternative locations of the Ctenophora (dashed gray line) and with Amoebozoa as an outgroup. Common names of exemplary species are in parentheses.
Fig. S5.
Fig. S5.
Summary of pocket protein and E2F/DP protein families in analyzed genomes. Rows represent different sequenced genomes, which have been organized from premetazoan (green) to metazoan (yellow and red). Columns denote the number of homologs discovered in each genome, where different paralog subgroups were classified using our pocket protein phylogeny (Fig. S6) and E2F/DP phylogeny (Fig. S7) (SI Materials and Methods). Gray rows denote changing names and column positions of paralogs that have arisen from gene duplication at the two major transitions (green to yellow and yellow to red). (Bottom) The gray row reflects the pocket protein and E2F/DP gene names in Homo sapiens.
Fig. S6.
Fig. S6.
Pocket protein phylogeny. Phylogeny depicts evolutionary relationships between 95 sequences of the pocket protein family in the metazoan and closely related lineages. Columns with the top 25% ZORRO score (980 positions) were used in our alignment. Confidence at nodes was assessed with multiple support metrics using different phylogenetic programs under the LG+I+G+F model of evolution (aBayes and SH-aLRT metrics with PhyML; RBS with RAxML). The tree from RAxML is shown, and colored dots at branches indicate corresponding branch supports. Thick branches indicate significant support by at least two metrics, one parametric and one nonparametric; branch support thresholds are shown in the center of the figure (SI Materials and Methods). For species abbreviations, refer to Fig. S5. aRB, ancestral RB.
Fig. S7.
Fig. S7.
E2F and DP protein phylogeny. Phylogeny depicts evolutionary relationships between 264 sequences of the E2F and DP protein family in the metazoan and closely related lineages. Columns with the top 10% ZORRO score (488 positions) were used in our alignment. Confidence at nodes was assessed with multiple support metrics using different phylogenetic programs under the JTT+I+G+F model of evolution (aBayes and SH-aLRT metrics with PhyML; RBS with RAxML). The tree from RAxML is shown, and colored dots at branches indicate corresponding branch supports. Thick branches indicate significant support by at least two metrics, one parametric and one nonparametric. Branch support thresholds are shown in the center of the figure (SI Materials and Methods). For species abbreviations, refer to Fig. S5. The fact that Petromyzon marinus E2F1236 forms a well-supported clade with E2F1, E2F2, and E2F3 suggests that the odd location of the E2F6 lineage outside of E2F1236 can be explained by what is called a long-branch attraction artifact (47). Nevertheless, improved sampling of the jawless fish and cartilaginous fish should clarify further the order of duplication events and support during this key evolutionary transition, particularly the relationships at the base of the E2F123 lineages. DEL (DP–E2F–like), ancestral E2F78 in plants.
Fig. 6.
Fig. 6.
Evolutionary model of sequences involved in E2FCM–pocket protein association. Family members are arranged according to their phylogenetic distances over their entire sequences. The VP*P motif in the E2F β3-strand, hydrophobic residues in RbCcore, and whether the RbCnter sequence is present are indicated. The amino acid number of the last residue in each human sequence is shown.
Fig. S8.
Fig. S8.
Alignment of pocket protein and E2F sequences. Sequences in the pocket protein C terminus around the structured core and RbCnter are shown. E2F sequences containing the β3-strand in the E2FCM domain are shown. Full organism names can be found in Fig. S5.
Fig. S9.
Fig. S9.
Sequence alignment of pocket protein sequences that show the emergence of K475 and H555. It has been demonstrated that K475 and H555 (human Rb numbering) contribute to the higher affinity of Rb for activator E2F transactivation domains compared with p107 (27). Alignment of sequences in the pocket protein pocket domains demonstrates that K475 and H555 first appear in the cartilaginous fish (Callorhinchus milii, Leucoraja erinacea, Squalus acanthias) but are maintained through the human sequence. Full organism names can be found in Fig. S5.

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