In contrast, the binding thermodynamic signature of AM-7209 was markedly influenced by mutations in the lid region (Figure ). Generally, weaker binding mutants exhibited a more negative entropy of binding and a more positive enthalpy of binding. Mutations predicted to have little-to-no effect on helix stability in the lid region (V14G, Q18G, E23G, E23R:R97E) showed entropies of binding similar to wild-type. Mutations predicted to disrupt helix formation (I19G, T16G:I19G) exhibited entropies of binding similar to the lid-truncated construct. The T16G mutant behaved intermediately between the two other classes of mutants. This supports the idea that binding of AM-7209 to weaker mutants is entropically favored due to the absence of lid ordering but enthalpically disfavored because of a reduced number of contacts between the ligand and the lid.