Taken together these results suggest that formation of a stable helical motif in residues 21-25 at the base of the MDM2 lid region requires additional contacts between the methyl groups of I19, the methyl group of T16 and the chlorophenyl ring of AM-7209 (Figure ). Disruption of this network of hydrophobic contacts increases lid flexibility and unwinds the helical motif. This explanation also accounts for the significant loss of affinity of AM-7209 for the T16G mutant observed in ITC experiments (Figure ). Thus, these results suggest that ordering of the MDM2 lid into a helical turn-extended motif is driven by shielding of a cluster of protein and ligand hydrophobic moieties, which is reminiscent of hydrophobic collapse observed in protein folding. To support this interpretation additional simulations were carried out with a modified version of AM-7209 in which the chlorophenyl moiety pointing towards T16 was replaced by a phenyl ring (AM-7209-Cl, Figure ). The decreased contacts between the ligand and T16 were observed to destabilise the helical motif in a manner qualitatively similar to the effects of the I19G mutation (Figure ). These findings provide an experimentally testable hypothesis for the proposed hydrophobic collapse mechanism.