Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Nature Publishing Group full text link Nature Publishing Group Free PMC article
Full text links

Actions

Share

.2015 Jul 2;523(7558):111-4.
doi: 10.1038/nature14405. Epub 2015 May 18.

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

Affiliations

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

Bradley A Webb et al. Nature..

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 requirements. Mutations inhibiting PFK1 activity cause glycogen storage disease type VII, also known as Tarui disease, and mice deficient in muscle PFK1 have decreased fat stores. Additionally, PFK1 is proposed to have important roles in metabolic reprogramming in cancer. 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-Mg(2+) 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 cancers, 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Activity of purified recombinant PFKP
a, Coomassie-stained SDS-PAGE of purified PFKP. The molecular weight (MW) of protein standards is shown in kilodalton (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 ± SEM of 10 (b) or 5 (c) determinations from 2 separate protein preparations.
Extended Data Figure 2
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 N-terminal (cyan) and the 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 7-stranded β-sheet surrounded by 6 α helices. Each SBD consists of 4-stranded β-sheet surrounded by 5 α helices. Two phosphate ions (stick drawings) are bound in pockets equivalent to the effector binding sites of theE. 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 modeled 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 modeled 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 vs relative enzymatic activity of PFKP in the presence (black triangles) and absence (grey circles) of 10 mM sodium sulfate. Activity is expressed relative to maximal activity at this pH and fructose 6-phosphate concentration. Data are means ± SEM of 3 determinations.
Extended Data Figure 3
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
Extended Data Figure 4. Structural comparison of ATP- and ADP-bound of PFKP with R- and T-state ofE. coli PFK
a. Structural overlay of ATP-bound (coloured) and ADP-bound (grey) of PFKP. For the sake of comparison with structures ofE. coli PFK, the N-terminal and C-terminal subdomains of PFKP are coloured cyan and blue for subunit A; yellow and orange for subunit B.b. The view in panela is slabbed so as to highlight the difference between the two structures.c. Structural overlay of R-state (coloured; PDB code: 4PFK) and T-state (grey; PDB code: 6PFK) ofE. coli PFK.d. The view in panelc is slabbed.
Extended Data Figure 5
Extended Data Figure 5. A unique tetramer interface in PFKP
a, Alignment of residues from PFKP surrounding Phe649 (arrow) with human PFKM and PFKL andSaccharomyces cerevisiae PFK1 alpha and beta subunits.b. Structure of PFKP tetramer.c. Structure ofScPFK tetramer, viewed roughly in the same orientation as PFKP. The tetramer interface is highlighted in the red box.d. Structure of rabbit PFKMe. Stereo drawing of the overlay of the tetramer interface of PFKP (in color) andScPFK.
Extended Data Figure 6
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 wild type PFKP (c) in buffer containing inhibitor (1mM citrate). Red arrows indicate dimers. Scale bar, 50 nm.d, Activity of wild type PFKP and PFKP-F649L in buffer containing 3 mM ADP, 3 mM ATP and 8 mM F6P. Data are means ± SEM of 6 (wild type) and 9 (F649L) determinations from 2 independent protein preparations (p < 0.001).
Extended Data Figure 7
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 3 experiments from individual preparations of cells.c, PFK1 activity (μmol F1,6bP produced per minute per ng of total cell lysate) was measured in 5 independent preparations of cells. A two-sided paired t-test was used to determine significance. ** P<0.01; ***P<0.001.
Figure 1
Figure 1. Structure of ATP-bound tetrameric PFKP is in the active conformation
a, TEM images of PFKP. Left panel – bar represents 50 nm. Right panel – Indicated dimensions are the mean ± SD of 37 individual particles.b-c, Ribbon diagrams of PFKP displaying the relative orientation of PFKP tetramer subunits. Each subunit is individually coloured. Arrows labeled “c” and “t” indicate the catalytic and tetramer interfaces, respectively. View in (c) is rotated 90° from (b).d, View rotated 90° from (c) displaying the catalytic sites. The front subunits are shown in ribbon representation and the rear subunits depicted as surface models. ATP, black; Mg2+, dark green; phosphate, yellow.e, The binding mode of ATP-Mg2+ at the active site of PFKP. The binding mode of the F6P substrate inScPFK is also shown.
Figure 2
Figure 2. PFKP undergoes a large conformational change upon ATP hydrolysis
a, Structural overlay of ATP-bound (cyan) and ADP-bound (grey) PFKP subunits.b, Structural overlay of ATP-bound (coloured ribbon) and ADP-bound (grey surface) PFKP tetramers. Arrows labeled “c” and “t” represent the catalytic and tetramer interfaces, respectively.c–d, Conformation of the active site in the ATP-Mg2+-bound (c) and ADP-bound (d) structures.
Figure 3
Figure 3. Interactions at the tetramer interface of PFKP regulate enzyme activity
a, Interface of the PFKP tetramer, with one of two tetramer interfaces involving residues from subunit A (cyan) and subunit D (magenta). Two views of the hydrophobic interactions at the tetramer interface and predicted electrostatic interactions between Arg613 of one subunit with Glu657 of the adjacent subunit. Arrows indicate the position of the two-fold symmetry axis in the tetramer interface, relating the two subunits.b, TEM images of wild type (WT) and PFKP-F649L PFKP particles in buffer containing activator and substrates or wild type in buffer containing the inhibitor citrate.c, Scatter plot of length vs width for particles observed in TEM. WT with activator and substrate (black, n=53); F649L with activator and substrate (blue, n=77); WT with citrate, tetramers (green, n=76) and dimers (red, n=41).d, F6P dependence of PFKP wild type (black squares) and E657A (grey circles) at 0.25 mM ATP. Data are means ± SEM of 8 (wild type) and 5 (E657A) determinations from 2 independent protein preparations.
Figure 4
Figure 4. Somatic cancer mutations of PFKP alter enzymatic activity and allosteric regulation
a–c, Location of indicated PFKP mutations in human cancers identified from the COSMIC database and mapped onto the catalytic interface of the PFKP subunit. Mutations chosen for further analysis are denoted with coloured boxes, including Arg48Cys (a, red) and location of Arg48 at PO42−-binding site (b), Asp564Asn (a, green) and ionic bond of Asp564 with Arg319 (b), and Asn426Ser (a, blue) and location of Ans425 at the catalytic interface (c).d–f, The effect of mutations on citrate inhibition (d), ATP activation and inhibition (e), and affinity for F6P (f). Data are means ± SEM of 7 (d), 5 (e), and 7 (f) determinations from 2 independent protein preparations. WT, black circles; Arg48Cys, red squares; Asn426Ser, blue triangles; Asn564N, green triangles.g, Lactic acid excretion (μmol lactate excreted per hour per μg of total cell lysate) from MTLn3 rat mammary adenocarcinoma cells expressing wild type and mutant PFKP-GFP. Data are means ± SEM of 4 experiments. * p < 0.05; ** p < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schöneberg T, Kloos M, Brüser A, Kirchberger J, Sträter N. Structure and allosteric regulation of eukaryotic 6-phosphofructokinases. Biol Chem. 2013;394:977–993. - PubMed
    1. Tarui S, et al. Phosphofructokinase deficiency in skeletal muscle. A new type of glycogenosis. Biochem Biophys Res Commun. 1965;19:517–523. - PubMed
    1. Getty-Kaushik L, et al. Mice Deficient in Phosphofructokinase-M Have Greatly Decreased Fat Stores. Obesity (Silver Spring) 2009 doi: 10.1038/oby.2009.295. - DOI - PMC - PubMed
    1. Yi W, et al. Phosphofructokinase 1 Glycosylation Regulates Cell Growth and Metabolism. Science. 2012;337:975–980. - PMC - PubMed
    1. Moreno-Sánchez R, et al. Phosphofructokinase type 1 kinetics, isoform expression and gene polymorphisms in cancer cells. J Cell Biochem. 2012;113:1692–1703. - PubMed

Publication types

MeSH terms

Substances

Associated data

Related information

Grants and funding

LinkOut - more resources

Full text links
Nature Publishing Group full text link Nature Publishing Group Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp