A λ-dynamics investigation of insulin and other A3 variant binding affinities to the insulin receptor.

Insulin is a clinical insulin variant where a conserved valine at the third residue on insulin's A chain (Val) is replaced with a leucine (Leu), impairing insulin receptor (IR) binding by 140-500 fold. This severe impact on binding from such a subtle modification has posed an intriguing problem for decades. Although experimental investigations of natural and unnatural A3 mutations have highlighted the sensitivity of insulin-IR binding to minor changes at this site, an atomistic explanation of these binding trends has remained elusive. We investigate this problem computationally using λ-dynamics free energy calculations to model structural changes in response to perturbations of the Val side chain and to calculate associated relative changes in binding free energy (ΔΔ). The Leu mutation and seven other A3 substitutions were studied in this work. The calculated ΔΔ results showed high agreement compared to experimental binding potencies with a Pearson correlation of 0.88 and a mean unsigned error of 0.68 kcal/mol. Extensive structural analyses of λ-dynamics trajectories revealed that critical interactions were disrupted between insulin and the insulin receptor as a result of the A3 mutations. This investigation also quantifies the effect that adding an A3 C atom or losing an A3 C atom has on insulin's binding affinity to the IR. Thus, λ-dynamics was able to successfully model the effects of subtle modifications to insulin's A3 side chain on its protein-protein interactions with the IR and shed new light on a decades-old mystery: the exquisite sensitivity of hormone-receptor binding to a subtle modification of an invariant insulin residue.