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 K d 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, the solution NMR structure of a doublytruncated Skp1 homodimer was determined. The dimer interface was mapped by intermolecular NOEs which identified ten contact amino acids. The resulting structure of the Skp1ΔΔ dimer has semi-parallel 2-fold symmetry with an interface that buries ~700 Å 2 of predominantly hydrophobic surface that partially overlaps with subsite-1 of the F-box interaction interface, explaining why only the Skp1 monomer binds FBPs. To confirm the model, Rosetta was used to identify amino acid substitutions that might disrupt that dimer, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. Skp1ΔF97E behaved as a monomer at concentrations up to 500 µM and actively bound a model F-box protein, mammalian Fbs1. Therefore, the dimeric state is not required for Skp1 to carry out a basic biochemical function, and access to the monomeric state is expected to facilitate structural studies of the mechanism of regulation of protist Skp1 by posttranslational prolyl hydroxylation and glycosylation.

Cargo competition for a dimerization interface stabilizes a protease adaptor inCaulobacter crescentus

“…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 (Pini, et al, 2015). Interestingly, recent structures show that the E3 ubiquitin ligase adaptor Skp1 buries its F-box interaction site upon dimerization (Kim, et al 2020), illustrating that masking of cargo binding sites by self-interactions is more generally found in biological systems. Not all protease adaptors share this mode of binding.…”