The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3

Abstract

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.

Structural analysis of a function-associated loop mutant of the substrate-recognition domain of Fbs1 ubiquitin ligase

“…A DNA fragment encoding the SBD of Fbs1 (residues 117-297) in which the six nonconserved regions 1 (142-145), 2-3 (156-168), 3-4 (174-179), 5-6 (215-222), 7-8 (239-252) and 9-10 (275-286) of Fbs1 were substituted with those of FBG3 (Fbs1 SBD loop-mutant 1) was designed with codons optimized for expression in Escherichia coli and synthesized by GeneArt (Life Technologies). The preparation of a second mutant SBD derived from Fbs1, in which the nonconserved regions 2-3 (156-168), 5-6 (215-220), 7-8 (239-252) and 9-10 (277-286) of Fbs1 were substituted with those of FBG3 (loop-mutant 2), has been described previously (Kumanomidou et al, 2010(Kumanomidou et al, , 2015. DNA fragments were cloned into the expression plasmid pET-15b.…”

“…DNA fragments were cloned into the expression plasmid pET-15b. The expression and purification of these recombinant Fbs1 SBD mutants has been described previously (Kumanomidou et al, 2015). Purified Fbs1 SBD loop-mutant 1 protein was concentrated to 3.6 mg ml À1 by ultrafiltration in 25 mM Tris-HCl pH 7.5, 1 mM dithiothreitol.…”

“…His-tagged protein pull-down assays were performed as described previously (Kumanomidou et al, 2015). In brief, 15 mg purified, His-tagged Fbs1 SBD was immobilized on 30 ml 99.9 (100.0) Multiplicity 3.7 (3.7) hI/(I)i 22.6 (4.7) R merge (%) 9.9 (43.6) R meas † (%)…”

“…However, carbohydrate-binding activity of FBG3 has not been detected (Yoshida et al, 2011). The carbohydrate-binding site of Fbs1 comprises a small hydrophobic pocket located at the top of a -sandwich, but the corresponding region of FBG3 exhibits a conformation different from that of Fbs1 (Mizushima et al, 2004(Mizushima et al, , 2007Kumanomidou et al, 2015). These conformational differences are caused by the distinct hydrogen-bond networks among the loops 2-3, 5-6, 7-8 and 9-10…”

“…# 2016 International Union of Crystallography (Kumanomidou et al, 2015). To investigate the structural differences between the loops of the SBDs of Fbs1 and FBG3 in detail, we determined the crystal structure of a mutant Fbs1 SBD in which the six nonconserved regions (1, 2-3, 3-4, 5-6, 7-8 and 9-10) in Fbs1 were substituted with those of FBG3.…”