A {lambda}-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 insulins A chain (ValA3) is replaced with a leucine (LeuA3), 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 {lambda}-dynamics free energy calculations to model structural changes in response to perturbations of the ValA3 side chain and to calculate associated relative changes in binding free energy ({Delta}{Delta}Gbind). The Wakayama LeuA3 mutation and seven other A3 substitutions were studied in this work. The calculated {Delta}{Delta}Gbind 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 {lambda}-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{delta} atom or losing an A3 C{gamma} atom has on insulins binding affinity to the IR. Thus, {lambda}-dynamics was able to successfully model the effects of subtle modifications to insulins 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. SIGNIFICANCE STATEMENTThis work addresses a decades-old question of how subtle modifications to insulins A3 side chain affects its binding affinity to the insulin receptor. {lambda}-Dynamics computed free energies of binding match experimental activity trends with high accuracy. Atomistic insights into hormone-receptor protein-protein interactions were obtained through a detailed investigation of {lambda}-dynamic trajectories. This work quantifies the effects of adding and removing atoms to insulins conserved A3 residue and identifies clear conformational preferences for insulin A3 residues when bound to the insulin receptor.