Fig. 4: SJ46421 promotes selective cooperative ternary complex formation with BRD3^BD2.

a Thermodynamic parameters for the indicated binary and ternary complexes. Data are the average +/− 1 s.d. from n = 2 experiments. Values for ΔG and ΔH are expressed in kcal/mol⁻¹. b Biochemical assay monitoring inhibition of KLHDC2 diGly protein substrate ubiquitylation by PROTAC-mediated ternary complex formation, performed as in Fig. 3f. SJ46421-BRD3^BD2 or SJ46421 alone (left panel) or SJ46423-BRD3^BD2 or SJ46423 alone (right panel) were incubated with neddylated CRL2^KLHDC2, prior to adding fluorescent ubiquitin-charged UBE2R2 (i.e. the pre-formed thioester-linked UBE2R2~ubiquitin intermediate) and diGly protein substrate. Effective PROTAC protein degraders promote ubiquitin transfer to BRD3^BD2 neo-substrate concomitant with loss of diGly substrate ubiquitylation. Samples were reduced when reaction was quenched by addition of SDS buffer. Loss of substrate ubiquitylation was quantified. Shown is representative panels from n = 2 independent experiments. c Quantification of loss of diGly substrate ubiquitylation from the gels in panel. b Data are the average from n = 2 independent experiments. d Sequence alignment of the second bromodomain from BRD2, BRD3, and BRD4. Residue boundaries of the domains are shown, and E344 from BRD3^BD2 is highlighted in red (top panel). Superposition of BRD2^BD2 (salmon; 3ONI.pdb) and BRD3^BD2 (chocolate; 3S92.pdb) to the structure of the VHL-MZL-BRD4^BD2 ternary complex (cyan, orange, and red respectively;5T35.pdb). G382, E344, and G386 from BRD2, BRD3, and BRD4 respectively that mediate cooperative ternary complex formation with VHL are shown in sticks (bottom panel). e Electrostatic surface representation of KLHDC2 highlghting potential basic patches for interaction with BRD3 E344 (left panel). Cartoon representation from (e) with relevant basic residues shown in sphere representation (right panel). f Thermodynamic parameters for the indicated ternary complexes between KLHDC2 and the indicated swap mutant BRD proteins and the R56A mutant of KLHDC2. Experiments were performed n = 2 times. Source data are provided as a Source Data file.