Biology subjects

Papa, J. E.

Publications and source records attributed to Papa, J. E..

3 recordsLinked to original sources

bioRxiv2026

Conformational Expansion Underlies the Evolutionary Emergence of Redox Sensitivity in Vertebrate Glucokinases

Glucokinase (GCK) catalyzes the first step of glycolysis in pancreatic {beta}-cells, where it functions as the bodys primary glucose sensor. GCK is extremely sensitive to oxidative inactivation, both in vivo and in vitro. This characteristic provides a mechanism to regulate GCK activity via alterations in the cellular redox environment. To understand the molecular and evolutionary origins of redox regulation, we characterized the sensitivity to oxidative inactivation of four extant GCKs and five ancestral GCKs produced from a recent phylogenetic analysis of the vertebrate family. We find that two invertebrate GCKs are significantly less sensitive to oxidative inactivation compared to their vertebrate counterparts. We also demonstrate that an ancestral GCK from chordates (cGCK) is insensitive to oxidative inactivation, whereas an ancestral GCK from early vertebrates (vGCK) displays a degree of redox responsiveness comparable to the extant human enzyme. The redox insensitive cGCK ancestor lacks cysteine residues at two positions, Cys230 and Cys461, that are conserved in all redox sensitive ancestral and extant enzymes. We find that installation of cysteines at these positions is insufficient to install redox sensitivity into cGCK. Our data demonstrate that the appearance of redox responsiveness in GCKs coincides with an expansion in the conformational landscape of the protein that occurred during early vertebrate evolution. These observations support a model in which the emergence of redox sensitivity required the ability to sample a unique super-open conformation, an event that also facilitated the emergence of two orthogonal GCK regulatory strategies, allosteric regulation by substrate glucose and an inhibitory interaction with the glucokinase regulatory protein.

biochemistry↗

bioRxiv2026

Structural Co-optation and Loss-of-function Underlie the Evolution of Regulatory Novelty in the Glucokinase Regulatory Protein

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, GKRPs 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.

biochemistry↗

bioRxiv2026

Evolution of Protein Regulation in the Vertebrate Glucose Sensor

Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the bodys glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzymes conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a de novo loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions. Significance StatementGlucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzymes conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface -- a structural spandrel resulting from prior conformational expansion -- was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.

biochemistry↗