Abstract C63: Structure of the Skp1-Fbx4 complex reveals a new class of the SCF ubiquitin ligase

Abstract Ubiquitin-mediated proteolysis governs the levels of many cellular proteins via a triple-enzyme cascade, involving ubiquitin-activating (E1), conjugating (E2) and ligating (E3) activities. The E3 ligases recruit both E2 and specific substrates and catalyze the final step in ubiquitination reaction. SCF complexes are the largest family of E3 ubiquitin-protein ligases that consist of Cul1, Rbx1, Skp1 and a member of the F-box protein family. F-box proteins are the substrate-recognition components of SCF; they interact with Skp1 through the N-terminal ∼40-residue F box motif, to recruit substrates through their variable C-terminal protein-protein interaction domains, including WD40 repeats (Fbxw subfamily), leucine-rich repeats (LRRs; Fbxl subfamily) and other different or unknown domains (Fbxo subfamily). Fbx4, which is the F-box protein responsible for the recognition of cell-cycle regulator cyclin D1 and telomeric DNA-binding protein Pin2, belongs to the Fbxo family that lack recognizable protein-protein interaction motif in their C-terminus. Reduced Fbx4 expression appears to be responsible, at least in part, for overexpression of cyclin D1 in human cancers. On the other hand, overexpression of Fbx4 in human cells promotes degradation of endogenous Pin2 and results in progressive telomere elongation. In order to define the fundamental structural properties of Fbx4, we have determined the crystal structure of the human Fbx4 protein in complex with Skp1 by single-wavelength anomalous dispersion method. The structure of the binary complex reveals that Skp1 and the F-box domain of Fbx4 retain the same overall fold as in other Fbxl and Fbxw structures determined, and that binding of the F box to Skp1 is very similar to that in other Skp1-F box complexes. Interestingly, the C-terminal domain of Fbx4 is projected away from Skp1 and F box, to adopt an α/β fold with a seven-stranded β sheet surrounded by seven α helices, which is distinct from LRR (in Fbxl) and WD40 (in Fbxw), reinforcing the notion that Fbx4 represents a new family of substrate-recognition subunits in SCF E3s. Moreover, mutagenesis experiments have revealed a potential substrate-binding site in Fbx4 C-terminal domain. Taken together, our structural and biochemical data provide a framework for understanding the molecular mechanism of substrate recognition by the SCFFbx4 ubiquitin ligase. Citation Information: Cancer Res 2009;69(23 Suppl):C63.