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Abstract
Skp1 is a homodimeric protein that recruits substrate receptor F-box proteins (FBP) to Cullin-1 as part of the Skp1/Cullin-1/FBP (SCF) complex. The SCF is a class of E3 ligases that conjugates substrates with ubiquitins and signals them for proteasomal degradation. E3 ligases therefore serve a role in controlling the proteome. In protists, the intrinsically disordered C-terminal domain of Skp1 undergoes an O2-sensing process resulting in the formation of an O-linked pentasaccharide. First characterized using Dictyostelium Skp1, NMR-supervised molecular dynamics (MD) simulations indicated that the glycan alters the surrounding ensemble of local conformers to be more conducive to FBP binding, in concordance with Skp1 interactome studies in cell extracts. Analysis of the Skp1 glycan from the unrelated parasite Toxoplasma led to the finding that the terminal disaccharide is distinctive and assembled by two glycosyltransferases (GTs) unrelated to the one from Dictyostelium. X-ray crystallographic and modeling studies of the terminal GT ortholog from another species, Pythium ultimum Gat1, rationalized how the Toxoplasma GT was a UDP-Gal:α-glucoside α3-galactosyltransferase specific for Skp1, rather than a UDP-Glc:α-glucoside α4-glucosyltransferase in glycogen synthesis as previously annotated. Despite the substitution of a glucose for a galactose in the fourth position, MD analysis indicated that both pentasaccharides were structurally compatible and alter Skp1 conformations in a similar manner. The convergent evolution represented by the GT switch supports the significance and specificity of the pentasaccharide structure. A previously described second function of the glycan, that it inhibits Skp1 homodimerization, was explored. Truncation studies of Dictyostelium Skp1 showed that the intrinsically disordered C-terminal domain is not required for dimerization. NMR analysis mapped the dimer interface onto FBP-binding subsite-1, which was confirmed by Rosetta-informed mutagenesis. The dimer configuration would place the C-terminal domains and their glycans within close proximity which suggests the potential for physical interactions. To address this possibility, NMR analysis of glycosylated Skp1 identified chemical shift differences of residues inferred to reside in the intrinsically disordered C-terminal domain. These findings reinforce dual roles for the Skp1 glycan in modulating O2-sensing by regulating the availability of monomeric Skp1 for FBP recruitment and Skp1 conformations that are receptive to bind FBPs.