To further characterize ternary complex binding thermodynamics, we next used isothermal titration calorimetry (ITC) by performing reverse titrations . First, in titrations of BRD4 N433F or N140F tandems (competent for BD1 or BD2 binding alone, respectively) into preformed SIM1:VCB complex we observed 2:1 stoichiometry, molar binding enthalpy of ΔH = -11.6 and -9.1 kcal/mol, and Kd = 1.2 and 0.12 µM, respectively (Fig. ). In contrast, titration of BRD4 WT BD1-BD2 under identical conditions displayed stoichiometry of 1:1, and a large negative binding enthalpy ( H = -20 kcal/mol) corresponding to the sum of BD1 and BD2 binding (Fig. ). To study ternary complex formation in a cellular context, we interrogated VHL binding to full-length BRD4 WT, N140F, or N433F mutations using kinetic NanoBRET assays . In these experiments, the ternary complex was rapidly formed with SIM1, but not with cis-SIM1 or MT1 (Fig. ). Ternary complex formation however was markedly reduced with BRD4 N140F (Fig. , middle) and almost abolished with N433F (Fig. ). These results confirm SIM1 utilizes both BD1 and BD2 for cellular ternary complex formation and suggest preferential binding to BD2 over BD1, consistent with the ITC results and what was found previously with MZ1 . Additional experiments showed a more robust and sustained ternary complex for BRD2 and BRD4 with VHL induced by SIM1 as compared to MZ1, while that with BRD3 did not appear to be as prolonged or stable (Extended Data Fig. ). Together, these data evidence SIM1 intramolecularly engages BD2 and BD1 to form a 1:1:1 ternary complex with VHL and BRD4.