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

Insulin Wakayama is a clinical insulin variant where a conserved valine at the third residue on insulin's A chain (Val A3 ) is replaced with a leucine (Leu A3 ), 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 A3 side chain and to calculate associated relative changes in binding free energy (ΔΔ G bind ). The Wakayama Leu A3 mutation and seven other A3 substitutions were studied in this work. The calculated ΔΔ G bind 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 δ atom or losing an A3 C γ 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.