Mapping the GCK-GKRP interaction landscape using deep mutational scanning by reverse two-hybrid screening

Glucokinase (GCK) regulates insulin secretion in the pancreas and glycogen synthesis in the liver. The best characterized interaction partner of GCK is the glucokinase regulatory protein (GKRP). GKRP is expressed in the liver where it regulates GCK activity and abundance. In this study, we used a library of GCK single amino acid substitutions and a reverse two-hybrid screen to characterize the impact of GCK variants on GKRP interaction. We obtained interaction scores for 7534 GCK single amino acid substitutions. A major driver of loss of interaction appeared to be loss of protein abundance, and we therefore compared interaction scores with our previously obtained GCK abundance scores to disentangle variant effects on interaction and abundance. Variants specifically perturbing interactions were found at the interaction interface but also structurally far from GKRP, revealing potential allosteric effects. In addition, unstable variants at buried residues in the large domain were stabilized by GKRP. These variants could potentially also be stabilized by e.g. pharmacological chaperones to rescue GCK function. In conclusion, we have now characterized the activity, abundance and interaction of 7128 GCK variants. Collectively these scores help pinpoint variants solely perturbing GKRP interaction, giving rise to liver-specific effects and potentially subtle glucose homeostasis phenotypes.