Extended Data Figure 7: Kinetic parameters for cereblon–DDB1–CC-885 binding to GSPT1, and sample electron density from the crystal structure of the complex.
From: A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase
a, Reference-corrected surface plasmon resonance binding curves for various concentrations of cereblon–DDB1 (threefold dilutions from 1 μM, coloured traces) flowed over a surface of covalently immobilized anti-GST antibody bound to GST–GSPT1 domains 2 and 3 at 10 °C in the presence of saturating levels of CC-885 or control compound glutarimide. Kinetic parameters shown were determined by fitting with a 1:1 kinetic binding model (black lines) using the Bioacore T200 kinetic analysis software package. Binding in the presence of gluatrimide could not be quantified. We show a representative set of curves from three independent experiments. For a 1:1 binding model where A + B ⇆ AB, the net rate of complex formation is given by the equation d[AB]/dt = ka[A][B] – kd[AB] and the rate of complex disassociation is given by kd[AB], where ka is the association rate constant (M−1s−1) and kd is the dissociation rate constant (s−1). KD, the equilibrium dissociation constant, is defined by KD = kd/ka. Rmax is a measurement of the analyte binding capacity, or maximum response. b, Analysis of the steady-state response versus the concentration of analyte in the presence of CC-885, and the determined affinity constant. c, GST–GSPT1 interacts with endogenous binding partner eRF1. We show that the purified GST–GSPT1 domains 2 and 3 (amino acids 437–633) protein used in this SPR binding assay and the electron miscroscopy experiments (Extended Data Fig. 8) is competent to bind purified eRF1 as reported in ref. 17. Coomassie stain, with lanes 1, 2 and 3 showing individual proteins. Lanes 4 and 5 show pull-down of GST; lanes 6 and 7 show pull-down of GST–GSPT1 domains 2 and 3, all were bound to magnetic glutathione beads incubated with eRF1 ± CC-885 for 1 h and washed three times. Lanes 8 and 9 show pull-down of GST-GSPT1 bound to magnetic glutathione beads incubated with CRBN–DDB1 ± CC-885 for 1 h and washed three times. This experiment was performed twice. For gel source data, see Supplementary Information Fig. 1. d, Sample electron density from the cereblon–DDB1–CC-885–GSPT1 crystal structure with cereblon residues shown in purple, GSPT1 residues shown in grey, and CC-885 shown in green. Refined 2Fo − Fc density is shown as a blue mesh contoured at 1.4σ. Fo − Fc difference density, shown as a green mesh contoured at 4σ, was generated by a single round of Refmac5 refinement calculated in the absence of GSPT1 residues 570–577.
