Skp1 Dimerization Conceals Its F-Box Protein Binding Site

Abstract

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a Kd of 2.5 µM as determined by sedimentation velocity studies, yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ~750 Å 2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite-1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations up to 500 µM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization..

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

“…The substantial inhibitory activity of mutant DdSkp1(P143A), is consistent with determinants for Skp1 recognition lying outside of the active site region ( 33 , 52 , 53 ). Free DdSkp1 has a well-ordered fold over its first 122 residues that is near identical to its structure in complex with F-box proteins ( 84 ), but evidence indicates that the C-terminal 40 residues, which include the hydroxylated Pro 143 in its central region and corresponds to the longest peptide tested, is disordered and subject to reorganization by glycosylation ( 38 , 85 ). Thus, the dynamics of substrate binding by DdPhyA and TgPhyA are likely different not only with respect to the PHDs but also with respect to bacterial orthologues, such as PPHD.…”

Cargo competition for a dimerization interface restricts and stabilizes a bacterial protease adaptor

“…The region mutated in our RcdA 3E variant may represent the binding interface for currently unknown adaptors that fulfill the role for PopA in other species where CtrA is degraded, such as Sinorhizobium meliloti (28). Recent structures show that the E3 ubiquitin ligase adaptor Skp1 buries its F-box interaction site upon dimerization (29), illustrating that masking of cargo binding sites by self-interactions can be generally found in biological systems.…”

The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii

“…pET-TEV_ Tgspy was electroporated into E. coli BL21-Gold (DE3), and its expression was induced in the presence of 0.5 mM IPTG for 18 h at 22 °C. Cells were collected and protein was extracted and purified over a Ni +2 -column and a Superdex 200 column, essentially as described previously for His 6 Skp1 ( 53 ). A highly enriched sample from an included volume fraction was analyzed.…”