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. 2019 Jun 24;9(1):9198.
doi: 10.1038/s41598-019-45704-z.

Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation

Xiaoying Zhang  1 , Qingya Shen  1 , Zhen Lei  1 , Qianyi Wang  1 , Jimin Zheng  2 , Zongchao Jia  3
Affiliations

Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation

Xiaoying Zhang et al. Sci Rep. .

Erratum in

Abstract

A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) The overall structure of AceK-Mn2+. The active site of AceK includes a buried ADP molecule and a bound AMP at the interface of the two domains. The kinase domain (KD) on the left resembles eukaryotic protein kinases. The regulatory domain (RD) on the right does not have any structural homologues. Loop-β3αC is coloured pink. (b) Structural comparison of AceK with Mn2+ (pink) and Mg2+ (light yellow). The location of metal ions is indicated by a red arrow.
Figure 2
Figure 2
Electron density (2mFo − DFc) map (contoured at 2σ, in cyan) at the catalytic center of AceK shows the two-metal center and coordinating residues. Anomalous density is shown in orange and contoured at 4σ. Manganese ions are represented as purple spheres.
Figure 3
Figure 3
Catalytic center of AceK. (a) The manganese ion (salmon pink) binding sites are shown in the current AceK structure with ADP bound. The loop-β3αC is highlighted in the structure, in red. Interactions between Mn2+ ion and the residues in the binding pocket are shown as dashed lines. (b) The magnesium-binding site (green) in the previous AceK structure with ATP bound. Interactions between metal ions and the residues in the binding pocket are shown as dashed lines.
Figure 4
Figure 4
Enzymatic activity assay of AceK with Mn2+/Mg2+. (a) Relative phosphatase activity of AceK at three different protein concentrations (from left to right: 3.0 μM, 1.5 μM and 0.75 μM) with 2.0 mM MgCl2 or 2.0 mM MnCl2. Kinase inhibitor/phosphatase activator cocktail contains 5.0 mM AMP and 5.0 mM pyruvate. (b) Relative phosphatase activity of AceK with different concentrations of Mg2+or Mn2+; error bars indicate the standard deviations. (c) Relative kinase activity of AceK at four different protein concentrations (from left to right: 1.5 μM, 3.0 μM, 4.5 μM and 6.0 μM) with 2.0 mM MgCl2 or 2.0 mM MnCl2, and 2.0 mM ATP. (d) Relative kinase activity of AceK with different concentrations of Mg2+ or Mn2+; error bars indicate the standard deviations.
Figure 5
Figure 5
Phosphatase activity assay of AceK mutants with Mn2+/Mg2+. (a) Comparison of relative AceK phosphatase activity among AceK WT and the indicated mutants. (b) Comparison of relative AceK phosphatase activity among AceK WT and the indicated mutants, in the presence of the kinase inhibitor/phosphatase activator cocktail (5.0 mM AMP and 5.0 mM pyruvate). The assays were performed in the 20 mM Hepes buffer pH 7.0 containing 2.0 mM Mn2+/Mg2+ and 2.0 mM ADP at 37 °C. Three independent replicates were performed in every experiment; error bars indicate the standard deviations.
Figure 6
Figure 6
ITC titration curves of Mn2+and Mg2+ binding to AceK WT in the presence of ADP or AMP. For all titrations, upper panel: the raw data, and lower panel: corresponding binding isotherm fitted according to a single set of identical sites model, and the solid lines represents the best fit. (a) Binding of Mn2+ to AceK WT in the presence of ADP and AMP. (b) Binding of Mg2+ to AceK WT in the presence of ADP and AMP. (c) Binding of Mn2+ to AceK WT in the presence of AMP. (d) Binding of Mg2+ to AceK WT in the presence of AMP. The concentration of ADP and AMP was both 0.25 mM.
Figure 7
Figure 7
ITC titration curves of Mn2+ and Mg2+ binding to mutant AceK in the presence of ADP and AMP. (a) Binding of Mn2+ to AceK D477A (b) Binding of Mg2+ to AceK D477A (c) Binding of Mn2+ to AceK D477K (d) Binding of Mg2+ to AceK D477K (e) Binding of Mn2+ to AceK D475A (f) Binding of Mg2+ to AceK D475A. The concentration of ADP and AMP was both 0.25 mM.
Figure 8
Figure 8
(a) The effects of metal ions on the phosphatase activities of AceK. All assays were performed in 20 mM Hepes, pH 7.0 buffer with 2.0 mM Mn2+. The concentration of metal ion was 2.0 mM. (b) Concentration dependence of the inhibition of AceK-catalyzed dephosphorylation of phospho-IDH by Cd2+. Three independent replicates were performed in every experiment; error bars indicate the standard deviations.
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