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, 42 (8), 597-603

Structural Study of Monomethyl Fumarate-Bound Human GAPDH


Structural Study of Monomethyl Fumarate-Bound Human GAPDH

Jun Bae Park et al. Mol Cells.


Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al . published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at 2.29 Å. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between NAD+bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the NAD+ cosubstrate due to steric hindrance of the nicotinamide portion of the NAD+ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

Keywords: crystallography; glyceraldehyde-3-phosphate dehydrogenase; inhibitor; monomethyl fumarate.

Conflict of interest statement


The authors have no potential conflicts of interest to disclose.


Fig. 1
Fig. 1. DMF inhibits the proliferation and function of human CD4+ and CD8+ T cells
CellTrace-Violet (CTV)-labelled human PBMCs were activated with 3 μg/ml soluble α-CD3 and 3 μg/ml soluble α-CD28 antibody in culture media alone or supplemented with the indicated doses of DMF for four days. (A) Representative flow cytometric plot of proliferating (CTVlow) cells that is gated on live CD4+ and CD8+ T cells. (B) Frequency of proliferating (CTVlow) CD4+ and CD8+ T cells as in A. (C) CTV profile showing proliferation versus IFN-γ gating on live CD4+ and CD8+ T cells. (D) Frequency of IFN-γ-secreting cells among live CD4+ and CD8+ T cells, as in C. Results are representative of three independent experiments. Data are shown as mean ± SEM and statistical significance was determined using a two-tailed unpaired Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig. 2
Fig. 2. Overall structure and MMF binding mode in the GAPDH active site
(A) The crystal structure shows that MMF-bound hGAPDH has a tetrameric structure based on the Q and R axes (the P axis is not presented). The dotted line represents the NAD+ binding site of hGAPDH. (B) The Fo-Fc electron density map (contour 1.0 σ) for covalently linked MMF with Cys152 of hGAPDH. (C) Comparison of the NAD+ binding site between NAD+-bound hGAPDH and MMF-bound hGAPDH. Two molecules are superimposed based on the C-α carbon of the protein backbone. (D) Two different binding modes of MMF in each protomer of tetrameric hGAPDH. Hydrogen bonds are shown as black lines.
Fig. 3
Fig. 3. Differences of binding modes to GAPDH between MMF and iodoacetate
(A) Chemical structure of iodoacetate and MMF. The red and purple round-squares show common and different chemical structures for both inhibitors, respectively. (B) The difference in inhibitory modes between iodoacetate and MMF.

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