A panel of eight mutants, selected on the basis of the MD simulations, was subjected to biophysical measurements to further investigate the relationships between predicted lid helix propensity, lid flexibility, and ligand binding energetics (Figures ). The compound Nutlin-3a, which does not order the intrinsically disordered lid of wild-type MDM2, served as a control. As anticipated, the ITC-derived thermodynamic signature of Nutlin-3a binding to MDM2 remained similar for all constructs, irrespective of the lid mutations (Figure ). This suggests that any interactions of Nutlin-3a with the lid, if present, are energetically inconsequential. This aligns with the notion that Nutlin-3a does not induce ordering of the MDM2 lid region upon binding.