Elucidating the complex allosteric regulation of PFK-1, a broadly evolutionarily conserved glycolytic enzyme

PROJECT SUMMARY/ABSTRACT Allosteric enzymes govern many metabolic and regulatory processes. Their regulation is modulated by the binding of an effector molecule to a site distal to their active site, inducing a conformational change that alters their activity. As such, the dysregulation of allosteric enzymes can cause metabolic disorders and cancer. However, despite its importance and ubiquity in biological processes, general mechanisms for protein allostery are poorly understood, and no single approach typically provides sufficient structural and energetic information to describe allosteric conformational changes in full molecular detail for most enzymes. Phosphofructokinase-1 (PFK-1) is a broadly conserved, allosterically regulated glycolytic enzyme. PFK-1 effectors elicit subtle conformational changes in the protein’s ensemble and oligomer state that modulate its activity. Cancer-associated somatic mutations in PFK-1 have been identified across the length of protein and in a wide range of cancer types. Altered PFK-1 allostery from somatic mutations and post translational-modifications has been shown to play a role in metabolic re-programming in cancer, making PFK- 1 an attractive therapeutic target. However, due to human PFK-1’s (hPFK) complex combinatorial regulation by ~20 effectors, for which few binding sites are known, structural data are insufficient for understanding shifts in hPFK’s conformational ensemble upon allosteric effector binding. This complex combinatorial regulation cannot be explained by a simple phenomenological model of allostery. A deeper understanding of hPFK’s ensemble regulation is necessary to determine how disease-associated mutations disrupt its allostery to drive disease phenotypes. A complete picture of the mechanism and evolution of combinatorial ligand regulation in hPFK will enable the rational design of allosteric therapeutics to specifically target this and other conserved, essential enzymes traditionally considered to be “undruggable”. In this proposal, I will train in multidisciplinary techniques, combining computational protein design, molecular dynamics (MD) simulations, traditional biochemistry, and hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to 1) discover the molecular basis for allosteric ligand regulation in hPFK, 2) map the evolution of allosteric ligand regulation in PFK-1 from prokaryotes to eukaryotes, and 3) design a peptide antibiotic that specifically inhibits multidrug-resistant (MDR) E. coli PFK-1. Our approach to discover the molecular details of PFK-1 effector regulation will enable future discoveries of how long-range allostery is encoded in other oligomeric enzymes. This work will deepen our understanding of the role of allosteric conformational changes to tune protein function in healthy and disease states.