Remarkably, analysis of the ITC-derived binding constants (Figure ) reveals that the affinity of AM-7209 for I19G is decreased by 1,000-fold compared to wild-type, comparable with the affinity measured for the lid-truncated construct. Conversely, mutations of residues that directly contact AM-7209 have a modest effect (V14G, Q18G) or intermediate effect (T16G) on binding constants. The significant effect of the T16G mutation on AM-7209 affinity suggests that the MD simulations may have underestimated the structural perturbation of the lid IDR caused by this mutation. The double mutant T16G:I19G binds AM-7209 with similar affinity to the single mutant I19G, suggesting that interactions of T16 with the ligand depend on formation of the helical motif in the lid. The importance of the E23-R97 salt bridge for lid ordering was probed with the E23G mutation. The resultant moderate loss of AM-7209 affinity is consistent with the moderate loss of helical propensity in MD simulations. Interestingly, swapping of salt-bridging residues in the E23R:R97E double mutant does not maintain wildtype binding affinity. This is consistent with MD simulations indicating a decreased propensity for formation of the R23-E97 salt bridge versus the native E23-R97 salt bridge. Uncertainties are the standard deviation of the mean from triplicate measurements.