An allosteric inhibitor of substrate recognition by the SCF(Cdc4) ubiquitin ligase

Abstract

The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF(Cdc4). A co-crystal structure reveals that SCF-I2 inserts itself between the beta-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 A away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

Figure 1

Small molecule inhibitors of the Cdc4-substrate interaction. a, Distribution of hits from 50,000 compound Maybridge library screen. Interaction between a fluorescein-labeled high affinity cyclin E-derived phosphopeptide (GLLpTPPQSG) and recombinant Cdc4 was monitored by fluorescence polarization. 44 compounds fell below the 50% inhibition cutoff (red line). Yellow dashed lines indicate three standard deviations above and below the mean. Z and Z’ factor scores were 0.8 and 0.66 respectively. At one standard deviation (σ), high controls were 4.6%, low controls 6.8%, and sample data 7.0%. b, Inhibition of interaction between full length phospho-Sic1 and Cdc4 (top panel). Phosphorylated Sic1 (0.1 μM) was incubated in the presence of recombinant Skp1-Cdc4 resin (500 ng) and the indicated compounds (50 μM). Bound protein was visualized by anti-Sic1 immunoblot. Total protein on resin after capture and wash was determined by Ponceau S stain (middle panel). DMSO solvent alone and 10 μM Gcn4 phosphopeptide (FLPpTPVLED) served as negative and positive controls. Inhibition of Sic1 ubiquitination in vitro (bottom panel). Phosphorylated Sic1 (0.2 μM) was incubated with recombinant SCF^Cdc4 (0.2 μM), E1 (0.4 μM), E2 (2 μM), ubiquitin (24 μM), and ATP (1 mM) in the presence of 80 μM indicated compound or control. Reaction products were visualized by anti-Sic1 immunoblot. c, Inhibition curves for SCF-I2 (red) and unlabeled CPD peptide (black) in the FP assay. (R) and (S) enantiomers of 1-(2-carboxynaphth-1yl)-2naphthoic acid (SCF-I2) are shown.

Figure 2

Structure analysis of the SCF-I2–Skp1-Cdc4 complex. a, SCF-I2 intercalates between β-propeller blades 5 and 6 of the Cdc4 WD40 repeat domain, approximately 25 Å from the CPD phosphopeptide binding site. SCF-I2 is shown in yellow. Red dot indicates modeled position of the P0 phosphate position. PB indicates propeller blade. b, Stereo diagram of SCF-I2 bound between PB5 and PB6 of the WD40 domain of Cdc4. SCF-I2 is shown in yellow; critical contact residues in Cdc4 are shown in blue stick representations. c, Surface representation of SCF-I2 binding region on Cdc4 in the absence (top) and presence (bottom) of bound SCF-I2. d, Stereo diagram of main chain conformational shifts induced by SCF-I2. The structure of Cdc4 in the absence of SCF-I2 but in the presence of a CPD phosphopeptide substrate (yellow) is shown in purple; the structure of Cdc4 in the presence of SCF-I2 (yellow) is shown in blue. e, Schematic of allosteric alterations caused by binding of SCF-I2. Positions of SCF-I2 bound conformations are shown in red; X indicates abrogation of an H-bond caused by rotation of Tyr574. f, Binding curves for WT Skp1-Cdc4 (black) and Skp1-Cdc4^Y574A (red) interactions with cyclin E-derived phosphopeptide by fluorescence polarization. g, SCF-I2 inhibition curves for WT Skp1-Cdc4 (black), Skp1-Cdc4^R655A (green) and Skp1-Cdc4^R664A (blue) binding to cyclin E phosphopeptide by FP. Inset shows binding inhibition by unlabeled cyclin E phosphopeptide for the same three proteins.

Figure 3

Inhibition and allosteric modulation of human WD40 domains. a, Fluorescence polarization competition binding curves for S. cerevisiae Cdc4 (black) and human Cdc4/Fbw7 (red) with SCF-I2. Inset shows inhibition by unlabeled cyclin E phosphopeptide for yeast Cdc4 (black) and human Fbw7 (red). b, Stereo view overlay of the inhibitor binding site region of S. cerevisiae Cdc4 (PDB 1NEX) in the absence of SCF-I2 (blue) with the corresponding region of human Cdc4/Fbw7 (green) (PDB 2OVR)^, . Only residues which differ between the human and S. cerevisiae proteins are labeled. c, Stereo view comparison of induced pockets in the WD40 repeat domain of Cdc4 and the bovine transducin Gβ subunit. Top displays a superposition of Cdc4 (blue) bound to SCF-I2 (yellow) with the G_Tβ subunit (dark green) bound to bovine retinal phosducin (pink) and a farnesyl ligand (magenta) from an associated G_Tγ subunit (PDB 1A0R). Bottom displays a superposition of unliganded forms of Cdc4 (grey) and the G_Tβ subunit (light green) (PDB 1TBG). For illustrative purposes, SCF-I2 and farnesyl ligands from the top image have been modeled into the lower image.