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

Nature volume 523, pages 111–114 (2015)Cite this article

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Abstract

Phosphofructokinase-1 (PFK1), the ‘gatekeeper’ of glycolysis, catalyses the committed step of the glycolytic pathway by converting fructose-6-phosphate to fructose-1,6-bisphosphate. Allosteric activation and inhibition of PFK1 by over ten metabolites and in response to hormonal signalling 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 proposed to have important roles in metabolic reprogramming in cancer4,5. Despite its critical role in glucose flux, the biologically relevant crystal structure of the mammalian PFK1 tetramer has not been determined. Here we report 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 characterize 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.

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Figure 1: Structure of ATP-bound tetrameric PFKP is in the active conformation.
Figure 2: PFKP undergoes a large conformational change upon ATP hydrolysis.
Figure 3: Interactions at the tetramer interface of PFKP regulate enzyme activity.
Figure 4: Somatic cancer mutations of PFKP alter enzymatic activity and allosteric regulation.

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Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

Data deposits

Structures of ATP–Mg2+- and ADP-bound PFKP have been deposited in the Protein Data Bank (PDB) under accession numbers 4XYJ and 4XYK, respectively.

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Acknowledgements

We thank S. Oakes for access to FPLC instrumentation, R. Fletterick and K. White for discussions, and A. Lauricella and G. DeTitta for the crystallization screening. This work was supported by National Institutes of Health R01 GM047413 to D.L.B., a Canadian Institutes of Health Research postdoctoral fellowship and a Pilot/Feasibility grant from the University of California, San Francisco, Liver Center (P30 DK026743) to B.A.W., and a grant from the Protein Structure Initiative of the National Institutes of Health U54-GM094597 to L.T.

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Author notes

  1. Bradley A. Webb and Farhad Forouhar: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Tissue Biology, University of California, San Francisco, 94143, California, USA

    Bradley A. Webb & Diane L. Barber

  2. Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, 10027, New York, USA

    Farhad Forouhar, Fu-En Szu, Jayaraman Seetharaman & Liang Tong

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  1. Bradley A. Webb
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Contributions

B.A.W. and D.L.B. conceived initial studies with recombinant PFKP. B.A.W. expressed and purified recombinant PFKP, and performed thermostability screens to identify buffer conditions for protein stability. Crystal screening hits were extensively optimized by F.E.S. and F.F. J.S. performed X-ray diffraction data collection and processing. F.F. determined and refined the structures. B.A.W. generated and biochemically characterized recombinant WT and mutant PFKP, and generated and analysed cells with heterologous PFKP expression. B.A.W., F.F., L.T. and D.L.B. contributed to writing the manuscript.

Corresponding authors

Correspondence to Liang Tong or Diane L. Barber.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Activity of purified recombinant PFKP.

a, Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of purified PFKP. The molecular mass (MM) of protein standards is shown in kilodaltons (kDa). b, Allosteric regulation of PFKP by ATP and ADP; F6P saturation curve PFKP in the presence of 0.25 mM ATP (filled black squares), 3 mM ATP (filled grey circles), 0.25 mM ATP and 0.25 mM ADP (open black squares), and 3 mM ATP and 3 mM ADP (open grey circles). c, Effect of ADP on kinetic behaviour of PFKP in the presence of 0.25 mM (black squares) or 3 mM (grey circles) ATP. Data in b and c are means ± s.e.m. of ten (b) or five (c) determinations from two separate protein preparations.

Extended Data Figure 2 Structure, nucleotide binding and phosphate ion binding of PFKP.

a, The structure of PFKP protomer can be divided into two halves: the amino (N)-terminal (cyan) and the carboxy (C)-terminal (yellow) subdomains. The N terminus of each subdomain begins with a nucleotide-binding domain (NBD) followed by a smaller substrate-binding domain (SBD). Each NBD closely resembles a canonical Rossmann fold composed of a seven-stranded β-sheet surrounded by six α-helices. Each SBD consists of a four-stranded β-sheet surrounded by five α-helices. Two phosphate ions (stick drawings) are bound in pockets equivalent to the effector binding sites of the E. coli PFK. b, Final 2Fo − Fc electron density at 3.1 Å resolution for ATP–Mg2+, contoured at 1σ. A strong electron density is observed near β- and γ-phosphate of the nucleotide, which was unambiguously modelled as Mg2+ ion. An extended but weaker electron density is also observed near the γ-phosphate of the nucleotide, which is surrounded by three backbone carbonyls of strictly conserved Ser32, Gly34 and Gly172. This electron density was modelled as a second metal ion, although it may belong to a water molecule. c, d, Structure of the two inorganic phosphate-binding sites in PFKP. e, Plot of concentration of ATP versus relative enzymatic activity of PFKP in the presence (black triangles) and absence (grey circles) of 10 mM sodium sulphate. Activity is expressed relative to maximal activity at this pH and F6P concentration. Data are means ± s.e.m. of three determinations.

Extended Data Figure 3 Structural comparison of the two PFKP tetramers in the ATP–Mg2+ complex.

a, Overlay of the structures of the eight PFKP subunits. Only two loops show substantial differences, indicated with the red arrows. b, Overlay of the two PFKP tetramers. A noticeable difference is the twisting of the second dimer in the two tetramers, indicated with the red arrow.

Extended Data Figure 4 Structural comparison of ATP- and ADP-bound PFKP with R- and T-state of E. coli PFK.

a, Structural overlay of ATP-bound (coloured) and ADP-bound (grey) PFKP. For comparison with structures of E. coli PFK, the N- and C-terminal subdomains of PFKP are coloured cyan and blue for subunit A, and yellow and orange for subunit B. b, The view in a is slabbed to highlight the difference between the two structures. c, Structural overlay of R-state (coloured; PDB accession number 4PFK)11 and T-state (grey; PDB accession number 6PFK)23 of E. coli PFK. d, The view in c is slabbed.

Extended Data Figure 5 A unique tetramer interface in PFKP.

a, Alignment of residues from PFKP surrounding Phe649 (arrow) with human PFKM and PFKL and S. cerevisiae PFK1 α- and β-subunits. b, Structure of PFKP tetramer. c, Structure of ScPFK tetramer15, viewed roughly in the same orientation as PFKP. The tetramer interface is highlighted in the red box. d, Structure of rabbit PFKM15. e, Stereo drawing of the overlay of the tetramer interface of PFKP (in colour) and ScPFK.

Extended Data Figure 6 Purification and TEM analysis of PFKP tetramer mutants.

a, Coomassie-stained SDS–PAGE of PFKP F649L and E657A. b, c, TEM images of PFKP F649L (b) in buffer with activator and substrates (3 mM ADP, 3 mM ATP and 8 mM F6P) and WT PFKP (c) in buffer containing inhibitor (1 mM citrate). Red arrows indicate dimers. Scale bar, 50 nm. d, Activity of WT PFKP and PFKP-F649L in buffer containing 3 mM ADP, 3 mM ATP and 8 mM F6P. Data are means ± s.e.m. of six (WT) and nine (F649L) determinations from two independent protein preparations (P < 0.001).

Extended Data Figure 7 Purification of PFKP cancer mutants and their activity in cells.

a, Coomassie-stained SDS–PAGE of purified recombinant PFKP mutants R48C, N426S and D564N. b, Immunoblot of GFP and actin from total cell lysates of MTLn3 rat mammary adenocarcinoma cells expressing PFKP–GFP. Blots are representative of three experiments from individual preparations of cells. c, PFK1 activity (micromoles of F1,6bP produced per minute per nanogram of total cell lysate) was measured in five independent preparations of cells. A two-sided paired t-test was used to determine significance. **P < 0.01; ***P < 0.001.

Extended Data Table 1 Data collection and refinement statistics
Full size table
Extended Data Table 2 Saturation kinetics on WT and mutant PFKP
Full size table
Extended Data Table 3 Somatic mutations of PFKP in cancer
Full size table

Supplementary information

Conformational change in PFKP upon ATP hydrolysis

The trajectory between the ATP-Mg2+-bound and the ADP-bound structures, viewed at the catalytic interface. (MOV 2172 kb)

Conformational change in PFKP upon ATP hydrolysis

The trajectory between the ATP-Mg2+-bound and the ADP-bound structures, viewed at the tetramer interface. (MOV 2830 kb)

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Webb, B., Forouhar, F., Szu, FE. et al. Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations. Nature 523, 111–114 (2015). https://doi.org/10.1038/nature14405

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