Fig. 3: Generation of a PROTAC employing KLHDC2.

a Structural superposition of KLHDC2^SBD (cyan, surface) bound to SJ46418 (orange, sticks) with the diGly substrate EPHB2 (chocolate, sticks, 8EBL.pdb). For clarity surface representation is depicted for the KLHDC2^SBD bound to SJ46418 only. Probable exit vectors from the ligands are depicted by arrows. Potential neo-substrate interaction surfaces with the KLHDC2^SBD U-shaped binding cleft is shown. b Chemical structure and biophysical characterization of SJ46418. Data are the average of n = 2 independent experiments. c Structural superposition of the KLHDC2^SBD (electrostatic, surface) bound to SJ46418 (orange, surface) to the diGly substrate EPHB2 (chocolate, surface, 8EBL.pdb) and the KLHDC2 targeting ligand KDRLZK1-KLHDC2^SBD (yellow, surface, 8SGE.pdb). For clarity surface electrostatic representation is depicted for KLHDC2^SBD bound to SJ46418 only. Probable exit vectors deriving from attachment sites to the ligands is depicted by arrows. d Chemical structure of JQ1 based KLHDC2 PROTAC protein degraders SJ46421 and SJ46423. e Fluorescent scan of gel monitoring SJ46421- and SJ46423-dependent ubiquitylation of the BD2 domain from BRD2, BRD3, or BRD4. Reactions were performed in pulse-chase format, with fluorescent ubiquitin charged-UBE2R2 added to neddylated CRL2^KLHDC2 and indicated PROTAC/BRD mixture. Shown is representative panels from n = 2 independent experiments. f Cartoon depiction of the biochemical assay monitoring inhibition of KLHDC2 di-Gly substrate ubiquitylation by PROTAC-mediated ternary complex formation. Briefly, neddylated CRL2^KLHDC2 was incubated with BRD3^BD2 and the indicated PROTAC prior to adding fluorescent ubiquitin-charged UBE2R2 (i.e. the pre-formed thioester-linked UBE2R2~ubiquitin intermediate) and a di-Gly protein substrate. Loss of di-Gly protein substrate ubiquitylation was quantified. g Morrison fit of data from competition ubiquitylation assay, yielding a Ki^app as a measure of the strength of ternary complexes promoted by SJ46421-BRD3^BD2 and SJ46423-BRD3^BD2. Data are the average from n = 2 independent experiments. Source data are provided as a Source Data file.