Structural mechanism for recognition of E2F1 by the ubiquitin ligase adaptor Cyclin F

Abstract

Cyclin F, a non-canonical member of the cyclin protein family, plays a critical role in regulating the precise transitions of cell-cycle events. Unlike canonical cyclins, which bind and activate cyclin-dependent kinases (CDKs), Cyclin F functions as a substrate receptor protein within the Skp1-Cullin-F box (SCF) E3 ubiquitin ligase complex, enabling the ubiquitylation of target proteins. The structural features that distinguish Cyclin F as a ligase adaptor and the mechanisms underlying its selective substrate recruitment over Cyclin A, which functions in complex with CDK2 at a similar time in the cell cycle, remain largely unexplored. We utilized single-particle cryo-electron microscopy to elucidate the structure of a Cyclin F-Skp1 complex bound to an E2F1 peptide. The structure and biochemical analysis reveal important differences in the substrate-binding site of Cyclin F compared to Cyclin A. Our findings expand on the canonical cyclin-binding motif (Cy or RxL) and highlight the importance of electrostatics at the E2F1 binding interface, which varies for Cyclin F and Cyclin A. Our results advance our understanding of E2F1 regulation and may inform the development of inhibitors targeting Cyclin F.

Figure 1.

A) Model for E2F1 ubiquitylation by SCF^CyclinF and proteasomal degradation. Cyclin F binds and activates the Skp1-Cul1-F box (SCF) E3 ligase to E2F1 and other substrates. B) Domain architecture for Cyclin F and Cyclin A. C) Final electron density map of E2F1-Cyclin F-Skp1 complex with local resolution from 3.0–4.0 Å, visualized by the color-coded surface. D) Final model of the E2F1-Cyclin F-Skp1 complex (PDB: 9CB3). E2F1 (pink), Skp1 (green), and Cyclin F (domains colored according to figure B) are shown. E) Overlay of E2F1-Cyclin F (9CB3) and E2F1-CDK2-Cyclin A (PDB: 1H24) cyclin domains. E2F1 peptide (pink), Cyclin A (green), CDK2 (yellow), Cyclin F cyclin domain (blue), and Cyclin F CBD (orange) are shown.

Figure 2.

A) Overlay of the E2F1-binding interface in Cyclin F and Cyclin A. The E2F1 peptide is from the E2F1-Cyclin F structure. B) Location of amino acids that are not conserved between Cyclin F and Cyclin A are rendered as sticks. C) Fluorescence polarization (FP) binding curves for Cyclin F-Skp1-GST and GST-Cyclin A for the TAMRA-E2F1 peptide. D) FP binding affinity values of alanine mutant Cyclin F proteins for TAMRA-E2F1 peptide. The Loop Mut contains alanine substitutions in the extended loop at all residues between D504-T511. E) HEK293T cells were co-transfected with plasmids encoding Flag-Cyclin F wild type (WT), a mutant M309A/L311A (MRYIL) mutant, or a mutant containing D504-T511 all changed to alanine (Loop Mut) along with a plasmid encoding Strep-E2F1. Anti-Flag antibodies were used for coimmunoprecipitation, and Western blots were performed with anti-Strep and anti-Flag antibodies to detect Strep-E2F1 and Flag-Cyclin F, respectively. F) HEK293T cells were transfected with plasmids encoding Flag-Cyclin F WT, the MRYIL, or Loop Mut. Coimmunoprecipitation with anti-Flag antibodies was followed by Western blot analysis to detect endogenous CP110 and Cdc6.

Figure 3.

A) SPOT peptide array of E2F1 and p130 peptide probed for GST-Cyclin F (25–546)-GST-Skp1 binding with an anti-GST antibody. B) Sequence alignment of RxL motifs from Cyclin F substrates. C) Fluorescence polarization competition assay with E2F1 RxL mutant peptides. D) Coulombic electrostatic surface potential for Cyclin F and Cyclin A at the E2F1 peptide binding interface. Numbers represent the fold change increase KD associated with the mutation in E2F1 peptides (from panel C).

Figure 4.

Model of SCF^CyclinF E3 ubiquitin ligase complex bound to E2F1-DP1. E2F1 (pink), DP1 (light blue) is positioned by Cyclin F (blue). Skp1 (green), Cul1 (yellow), and Rbx1 (grey) are shown.