GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

Abstract

Summary How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GID SR4 , which resembles an organometallic supramolecular chelate. The Chelator-GID SR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Cryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylation

“…Although at an overall level, Gid12-bound Gid4 superimposes with prior structures without Gid12 (0.47 Å RMSD, 6SWY.PDB; and 0.56 Å RMSD, 7NS3.PDB) (Fig. 3a ), these structures themselves show variability in the conformations of loops at the entrance to the substrate-binding tunnel 21 , 22 . Among the differences, Gid4’s L2-loop was not visible in the cryo-EM density for GID SR4 in the absence of substrate, potentially reflecting intrinsic conformational flexibility (Fig.…”

“…Reasoning that challenges in producing an individual protein can arise from exposure of a hydrophobic surface that is normally buried within a multiprotein complex, we tested if Gid12 could be produced upon co-expression with interaction partners. We generated individual baculoviruses for Gid12, all Gid subunits, and Moh1 (the yeast ortholog of human protein YPEL5, which binds the human Gid7 ortholog WDR26 22 , 24 ), some with GST or Strep affinity tags. Affinity purifications from lysates of insect cells infected with various combinations tested roles of each subunit in binding Gid12 and led to three key conclusions (Fig.…”

“…In agreement with the biochemical data, Gid12 primarily contacts Gid4, and also Gid5. Prior structures showed that Gid4 contains a cylindrical 8-stranded β-barrel domain that engages a substrate’s Pro/N-degron in a central tunnel 22 , 26 , while the exterior of the cylinder nestles in a semicircular groove formed by the SPRY domain of Gid1 and the C-terminal half of Gid5’s armadillo repeats 21 . Gid12 emanates from and extends this groove, to wrap ≈305° around Gid4.…”

“…However, another subunit, Gid7, substantially potentiates ubiquitylation and degradation of Fbp1 by driving supramolecular assembly of two GID SR4 E3s into a hollow oval structure termed Chelator-GID SR4 22 . Fbp1 is encapsulated in the center of the oval, much like a small ligand captured by an organometallic supramolecular chelate 22 .…”

“…When recombinant Gid7 is added to a GID SR4 complex (including two Gid1 and Gid8 subunits), size exclusion chromatography indicated multiple potential higher-order assemblies. Moreover, two human Gid7 orthologs form related but different higher-order assemblies 22 , 23 , raising the possibility that additional yeast Gid7-driven supramolecular assemblies remain to be discovered.…”