6v88

Publication Abstract from PubMed

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 muM 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 (Skp1DeltaDelta). The solution structure of the Skp1DeltaDelta dimer reveals a 2-fold symmetry with an interface that buries approximately 750 A(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 (Skp1DeltaF97E) behaved as a stable monomer at concentrations of </=500 muM 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, Skp1DeltaF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

Skp1 Dimerization Conceals Its F-Box Protein Binding Site.,Kim HW, Eletsky A, Gonzalez KJ, van der Wel H, Strauch EM, Prestegard JH, West CM Biochemistry. 2020 Apr 13. doi: 10.1021/acs.biochem.0c00094. PMID:32227851^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.