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Abstract
Skp1/Cullin1/F-Box protein (SCF) complexes represent a premier class of E3 ubiquitin ligases responsible for proteomic control by targeting proteins for degradation by the 26S proteosome. Substrate specificity is mediated by a large variety of F-box proteins (FBPs) which interact with the SCF complex via Skp1. Skp1 is utilized for oxygen sensing within the protist kingdom via oxygen-dependent glycosylation on its partially disordered C-terminal region (CTR). In the presence of oxygen, the prolyl hydroxylase PhyA hydroxylates Skp1 allowing for the attachment of a pentasaccharide by glycosyltransferases with absolute specificity for Skp1. Glycosylation of the CTR alters Skp1’s FBP binding repertoire in cells and inhibits its homodimerization which may sequester it from SCF complexes. However, a biochemical understanding of these effects is needed to interpret cellular consequences. Here, we establish in vitro systems where the effects of glycosylation can be quantified. Utilizing sedimentation velocity experiments (SV), we demonstrate that glycosylation weakens homodimerization of Skp1 from the parasite Toxoplasma gondii by an order of magnitude, an effect reproduced by CTR deletion. Replacing Skp1’s CTR with scrambled or poly-serine sequences indicate that amino acid composition is more important than sequence for CTR mediated homodimerization. The effects of high salt and molecular dynamics simulations indicate the presence of an electrostatically mediated fuzzy protein interaction of Skp1’s CTR that is interrupted by the glycan. Comparative studies with an unrelated protist, Dictyostelium discoideum, provided an example of evolutionary tuning of this mechanism. Gat1, Skp1’s terminal glycosyltransferase in Toxoplasma is detected in the Skp1 interactome based on co-immunoprecipitation, suggesting a non-enzymatic role for Gat1. SV indicates that Gat1 forms a direct complex with Skp1 which competes with homodimerization and whose stoichiometry is dictated by glycosylation. Computational modeling suggests a mechanism whereby Gat1 utilizes Skp1’s fuzzy interactions to dissociate the Skp1 homodimer, providing a second mechanism to regulate the availability of Skp1 to the SCF complex. Finally, we engineered recombinant expression systems for soluble F-box protein production for testing the effects of glycosylation on FBP binding. Overall, the studies reveal a novel mechanism for the regulation of E3(SCF) ubiquitin ligases involving titration of Skp1 availability for SCF complex formation.