The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.

SummaryThe elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.

The retinoblastoma proteinC-terminal domain (RbC) is necessary for the tumor suppressor protein's activities in growth suppression and E2F transcription factor inhibition. Cyclin-dependent kinase phosphorylation of RbC contributes to Rb inactivation and weakens the Rb-E2F inhibitory complex. Here we demonstratetwo mechanisms for how RbC phosphorylation inhibits E2F binding. We find that phosphorylation of S788 and S795 weakens the direct association between the N-terminal portion of RbC (RbCN) and the marked-box domains of E2F and its heterodimerization partner DP. Phosphorylation of these sites and S807/S811 also induces an intramolecular association between RbC and the pocket domain,which overlaps with the site of E2F transactivation domain binding. Areduction in E2F binding affinity occurs with S788/S795 phosphorylation that is additive with the effects of phosphorylation at other sites, and we propose a structural mechanism that explains this additivity. We find that different Rb phosphorylation events have distinct effects on activating E2F family members, which suggests a novel mechanism for how Rb may differentially regulate E2F activities.

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.cell cycle | tumor suppressor protein | E2F | protein-protein interactions | evolution E 2F transcription factors regulate the mammalian cell cycle by controlling expression of genes required for DNA synthesis and cell division (1). E2F activity is regulated by the retinoblastoma (Rb) "pocket" protein family members Rb, p107, and p130, which bind and inhibit E2F and recruit repressive factors to E2F-driven promoters (2-5). Beyond cell-cycle regulation, these pocket protein-E2F complexes are the focal point of signaling pathways that trigger diverse cellular processes including proliferation, differentiation, apoptosis, and the stress response. Improper inactivation of pocket proteins is a common mechanism by which cancerous cells maintain aberrant proliferation (1, 5-7). Pocket protein-E2F dissociation and subsequent E2F activation is induced by cyclindependent kinase phosphorylation (3-5, 8) or binding of viral oncoproteins such as the SV40 T-antigen (9).The E2F family contains eight members, five of which (E2F1 to E2F5) form complexes with pocket proteins (1). E2F1 to E2F3 associate exclusively with Rb and are potent activators of transcription during the G1 and S phases of the cell cycle (10, 11). These "activating" E2Fs also specifically induce apoptosis (12). E2F4 is found in complexes with all three pocket proteins and is thought of primarily as a repressor, because it typically occupies promoters of repressed genes and is exported from the nucleus upon release from pocket proteins (1,13,14). In contrast, several studies of E2F4 function during development suggest that E2F4 may stimulate proliferation in certain contexts, acting through association with other transcription factors (14). Better characterization of how E2F4 and E2F5 associate with pocket proteins and other factors is needed to unde...

The human pocket proteins Rb, p107 and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie why Rb is a uniquely potent tumor suppressor. It has been proposed that different E2F transcription factor binding partners mediate distinct pocket protein activities. In humans, Rb binds E2F1-5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin dependent kinase (Cdk) on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.