Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations

Abstract

Phosphofructokinase-1 (PFK1), the “gatekeeper” of glycolysis, catalyses the committed step of the glycolytic pathway by converting fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate. Allosteric activation and inhibition of PFK1 by over 10 metabolites and in response to hormonal signaling fine-tune glycolytic flux to meet energy requirements1. Mutations inhibiting PFK1 activity cause glycogen storage disease type VII, also known as Tarui disease2, and mice deficient in muscle PFK1 have decreased fat stores3. Additionally, PFK1 is suggested to have important roles in metabolic reprograming in cancer4,5. Despite its critical role in glucose flux, the biologically relevant crystal structure of the mammalian PFK1 tetramer has not been determined. We report here the first structures of the mammalian PFK1 tetramer, for the human platelet isoform (PFKP), in complex with ATP-Mg2+ and ADP at 3.1 and 3.4 Å, respectively. The structures reveal substantial conformational changes in the enzyme upon nucleotide hydrolysis as well as a unique tetramer interface. Mutations of residues in this interface can affect tetramer formation, enzyme catalysis and regulation, indicating the functional importance of the tetramer. With altered glycolytic flux being a hallmark of cancers6, these new structures allow a molecular understanding of the functional consequences of somatic PFK1 mutations identified in human cancers. We characterized three of these mutations and show they have distinct effects on allosteric regulation of PFKP activity and lactate production. The PFKP structural blueprint for somatic mutations as well as the catalytic site can guide therapeutic targeting of PFK1 activity to control dysregulated glycolysis in disease.