Structural basis of the phosphorylation-independent recognition of cydin D1 by the SCFFBXO31 ubiquitin ligase

Ubiquitin-dependent proteolysis of cydin D1 is associated with normal and tumor cell proliferation and survival. The SCFFBXO31 (Skp1-Cul1-Rbx1-FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cydin D1 degradation. Previous studies have suggested that cydin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm. Here we present the crystal structures of the Skp1-FBXO31 complex alone and bound to a phosphorylated cydin D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding domain consisting of two beta-barrel motifs, whereas cydin D1 binds to FBXO31 by tucking its free C-terminal carboxylate tail into an open cavity of the C-terminal FBXO31 beta-barrel. Biophysical and functional studies demonstrate that SCFFBXO31 is capable of recruiting and ubiquitinating cydin D1 in a phosphorylation-independent manner. Our findings provide a conceptual framework for understanding the substrate specificity of the F-box protein FBXO31 and the mechanism of FBXO31-regulated cydin D1 protein turnover.