Santiago, J. I.; Freye, C.; Kamalaldinezabadi, S. S.; Papa, J. E.; Whittington, A. C.; Miller, B. G.

The glucokinase regulatory protein (GKRP) derives from an ancestral etherase. Despite existing as a single locus in the metazoans, GKRP evolved multiple novel functions unrelated to etherase activity. In jawed vertebrates, a protein-protein interaction (PPI) emerged that inhibits glucokinase (GCK) activity in the liver. This PPI is critical to maintaining glucose homeostasis. In mammals, GKRP is allosterically regulated by carbohydrates, with 6-phospharylated sugars promoting inhibition of GCK by GKRP, while 1-phosphorylated sugars relieve inhibition. Here, we use a vertical evolutionary approach to identify the genetic, biochemical, and biophysical mechanisms underlying the emergence of small-molecule allostery in GKRP. We pinpointed a single leucine to valine substitution in the N-terminus of GKRP from the ancestor of the euarchontoglires that, when introduced into the non-regulated placental mammal GKRP ancestor, installed sensitivity to sorbitol-6-phosphate (S6P). Interestingly, GKRP's inhibitory activity in the absence of S6P was reduced but unchanged in its presence. The mutation enabled co-optation of the ancestral etherase active site, which also existed as an ambiguous phosphorylated carbohydrate binding site in unregulated GKRPs. This substitution likely introduced an alternative conformation of the N-terminus causing apo-GKRP to sample a binding incompetent state prior to GCK binding. Our results suggest a simple model of the evolution of protein functional novelty where a single mutation can cause a large functional shift via co-optation of pre-existing structural features. Importantly, in contrast to many models of protein evolution, ours does not require the addition of new genetic material to realize a novel function such as small-molecule allosteric regulation.