Structural basis of dimerization-dependent ubiquitination by the SCF(Fbx4) ubiquitin ligase

Abstract

The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F- box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF(Fbx4) (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adopts a compact alpha/beta fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

FIGURE 1.

Oligomerization state and ubiquitination activity of the Skp1-Fbx4 complexes. A, overlay of gel filtration chromatography profiles of Skp1-Fbx4^FL and Skp1-Fbx4^core. The retention volumes of proteins of known mass and the void volume of the Superdex 200 column are indicated. mA₂₈₀, milliunits at A_{280 nm}. B, representative sedimentation equilibrium data for the Skp1-Fbx4^FL (left, 100 μm and 12 krpm) and Skp1-Fbx4^core (right, 100 μm and 10 krpm). MW, molecular weight; Calc, calculated; krpm, kilorevolutions per minute. C, in vitro ubiquitination of Pin2^FL by Skp1-Fbx4^FL and Skp1-Fbx4^core in the presence and absence of αB-crystallin. Pin2 and Pin2-ubiquitin conjugates were detected by immunoblotting (IB) with an anti-Pin2 antibody. Ubn, more than two Ub molecules attached. D, time course of ubiquitination of the ³²P-labeled Pin2^FL protein in the absence and presence of Skp1-Fbx4^FL or Skp1-Fbx4^core. ³²P-labeled Pin2^FL and Pin2^FL-ubiquitin conjugates were analyzed by SDS-PAGE followed by autoradiography. HMW, high molecular weight. E, quantitative PhosphorImager analysis of the high molecular weight ubiquitinated Pin2^FL from D. The ratios of the high molecular weight Pin2/total Pin2 proteins are plotted as a function of reaction time. Data represent the mean ± S.D. for 2–3 independent experiments.

FIGURE 2.

Overall structure of the Skp1-Fbx4^core complex. A, ribbon diagram of the Skp1-Fbx4^core dimer. Fbx4^core and Skp1 are shown in red and blue in one molecule and in pink and cyan in the other one. B, ribbon diagram of the Skp1-Fbx4^core monomer. The secondary structure elements are labeled. Dotted lines represent disordered regions. The strands in Fbx4^core involved in dimerization are colored pink. Deletions of the NTD and finger are indicated by arrows. C, the topology diagram of the Fbx4^core dimer. α helices and β strands are shown in red and maroon in one molecule and violet and pink in the other one. Dotted lines represent disordered regions.

FIGURE 3.

Structure of the Fbx4 C-terminal domain is similar to that of small GTP-binding proteins. Superimposition of the Fbx4 C-terminal domain (red) and ARF1 (cyan; Protein Data Bank (PDB) code 1HUR) is shown. The unique N-terminal α helix of ARF1 is omitted for clarity.

FIGURE 4.

Dimer interface in the Skp1-Fbx4^core structure. A, a stereo view of the dimer interface of Fbx4^core. The two protomers are colored red and pink, respectively. Hydrogen bonds are represented by green dashed lines. The H5 and H13 helices are omitted for clarity. B, reciprocal affinity pulldown assays with the co-expressed His₆-F-box-linker-Skp1 complex and GST-G domain. Cell lysates were incubated with Ni²⁺-NTA and glutathione-Sepharose 4B (GS4B) beads, eluted with imidazole and/or glutathione, respectively, and analyzed with SDS-PAGE and Coomassie Blue staining. Purified GST protein was incubated with purified His₆-F-box-linker-Skp1 complex and used as the negative control. C, time course of ubiquitination of the ³²P-labeled Pin2^FL protein by three Fbx4^core deletion mutants. ³²P-labeled Pin2 and Pin2-ubiquitin conjugates were analyzed by SDS-PAGE followed by autoradiography. Ubn, more than two Ub molecules attached. D, time course of ubiquitination of the ³²P-labeled Pin2^FL protein by two Fbx4^core Ala mutants. ³²P-labeled Pin2 and Pin2-ubiquitin conjugates were analyzed by SDS-PAGE followed by autoradiography.

FIGURE 5.

Both the NTD and the finger regulate Fbx4 activity and dimerization. A, overlay of gel filtration chromatography profiles of Skp1-Fbx4^FL, Skp1-Fbx4^ΔNTD, Skp1-Fbx4^ΔFinger, and Skp1-Fbx4^core. The retention volumes of proteins of known mass are indicated. mA₂₈₀, milliunits at A_{280 nm}. B, Ni²⁺-NTA affinity pulldown assay characterizing the interaction of the His₆-tagged Pin2^NTD with the Skp1-Fbx4 complexes. Indicated proteins were incubated, and reactions were precipitated with Ni²⁺-NTA resin. The unbound (U) and eluted bound (B) fractions were analyzed by SDS-PAGE and Coomassie Blue staining. C, superimposed gel filtration profiles of the Skp1-Fbx4-Cul1-Rbx1 (CR) mixtures. The Skp1-Fbx4 complex (Skp1-Fbx4^FL, Skp1-Fbx4^ΔNTD, Skp1-Fbx4^ΔFinger, or Skp1-Fbx4^core) was incubated with Cul1-Rbx1 at a 1.5:1 molar ratio before loading onto the gel filtration column. The retention volumes of the four protein complexes and the individual subcomplexes are indicated. D, time course of ubiquitination of the ³²P-labeled Pin2^FL protein by Fbx4^FL, Fbx4^ΔNTD, Fbx4^ΔFinger, and Fbx4^core. ³²P-labeled Pin2 and Pin2-ubiquitin conjugates were analyzed by SDS-PAGE followed by autoradiography. Ubn, more than two Ub molecules attached.

FIGURE 6.

Models for dimerization-dependent ubiquitination by Fbx4^core and Fbx4^FL. A, the dimeric Skp1-Fbx4^core complex can directly interact with both the substrate (Sub) and Cul1-Rbx1 to promote the ubiquitination of the substrate. B, the monomeric Skp1-Fbx4^FL complex cannot bind substrate and Cul-Rbx1. It is possible that phosphorylation (circled P) on the Fbx4 NTD induces the conformational changes on both the NTD and the finger, thus enabling Fbx4 to dimerize and to interact with the substrate and Cul1-Rbx1.