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.

The Glycolytic Enzyme Phosphofructokinase-1 Assembles into Filaments

“…This arrangement resulted in different assembly interactions for each of the two dimers that make up the PFKL tetramer, which we refer to as dimers A and B. Although the overall approximate D2 symmetry of the PFKL tetramer remained intact, the resolution of our structure was insufficient to visualize small conformational differences in dimers A and B that may arise from their unique packing environments in the polymer; such asymmetry, however, would be consistent with asymmetry observed in PFKP crystal structures (Webb et al, 2015). The asymmetry in assembly interfaces between the A and B dimers of PFKL was unusual among helical filaments with multimeric helical asymmetric units.…”

“…We recently reported the first biologically relevant tetrameric mammalian PFK1 structure using recombinant platelet PFK1 (PFKP) produced using a baculovirus expression system (Webb et al, 2015). We used the same system to express and purify the liver and muscle isoforms of PFK1.…”

“…DNA primers were designed using an online primer design tool (Agilent Technologies) and were purchased from Elim Biopharmaceuticals. Cloning of human PFKP was previously described (Webb et al, 2015). The chimera of PFKP and PFKL was generated by Gibson Assembly (New England Biolabs, Inc.).…”

Tarui´s disease: Review and bioinformatic perspectives

“…El inadecuado funcionamiento de la FFQ también conlleva al depósito o almacenamiento excesivo de glucógeno intracelular, lo cual provoca compresión y desplazamiento de la maquinaria contráctil de las fibras musculares, con las manifestaciones clásicas de la enfermedad a nivel muscular (18). Sin embargo, la FFQ no solamente es de relevancia en esta enfermedad metabólica, sino que en conjunto con otras enzimas, ha sido estudiada en otros tipos de padecimientos como lo son la obesidad, diabetes y cáncer (19), por lo que su deficiencia también puede inducir padecimientos paralelos en los pacientes.…”

“…Además, en otras posibles líneas de modulación, se ha reportado que la activación y la inhibición alostérica de la enzima por más de diez metabolitos y en respuesta a la señalización hormonal resultan en la regulación del flujo glucolítico para satisfacer las necesidades de energía de las células (19 incluyen ATP, lactato, AMP, cAMP, ADP, fructosa-2,6-bisfosfato, citrato y acil-CoA (35) y se ha demostrado que estos moduladores alostéricos ejercen propiedades estimulantes o inhibitorios que interfieren con el equilibrio entre dímeros y tetrámeros de la enzima, lo cual hace parecer plausible que moléculas como el ATP, el citrato y el lactato inhiban la FFQ mediante la estabilización de su conformación dimérica, mientras que ADP, AMP, cAMP y fructosa-2,6-bisfosfato favorecerían la formación de tetrámeros (37).…”