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Crystal structures of human choline kinase isoforms in complex with hemicholinium-3: single amino acid near the active site influences inhibitor sensitivity.

Hong BS, Allali-Hassani A, Tempel W, Finerty PJ, Mackenzie F, Dimov S, Vedadi M, Park HW - J. Biol. Chem. (2010)

Bottom Line: This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays.Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta.Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKalpha (alpha1 and alpha2) and ChoKbeta isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKalpha1 and ChoKbeta isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKalpha1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKbeta was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

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HC-3 phosphorylation by ChoK. A, mass spectra of the supernatant from a reaction mixture of ΔN-ChoKβ in the presence of ADP. The supernatant was incubated for 15 min and contains the major peak at m/z 413.2, corresponding to native HC-3 (top), whereas the spectrum from the same reaction mixture incubated for 5 h reveals the presence of another peak at m/z 493.2 corresponding to Pho-HC-3 (bottom). B, HC-3 phosphorylation activity by ChoK isoforms. The experiments were performed using full-length wild-type ChoKα1 (○), ChoKα2 (▾), and ChoKβ (·) in the presence of [γ-32P]ATP. Data shown represent the means of triplicate determinations, and error bars indicate the standard deviations.
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Figure 5: HC-3 phosphorylation by ChoK. A, mass spectra of the supernatant from a reaction mixture of ΔN-ChoKβ in the presence of ADP. The supernatant was incubated for 15 min and contains the major peak at m/z 413.2, corresponding to native HC-3 (top), whereas the spectrum from the same reaction mixture incubated for 5 h reveals the presence of another peak at m/z 493.2 corresponding to Pho-HC-3 (bottom). B, HC-3 phosphorylation activity by ChoK isoforms. The experiments were performed using full-length wild-type ChoKα1 (○), ChoKα2 (▾), and ChoKβ (·) in the presence of [γ-32P]ATP. Data shown represent the means of triplicate determinations, and error bars indicate the standard deviations.

Mentions: Given the possibility that ΔN-ChoKβ is capable of phosphorylating HC-3, additional experiments were performed to verify whether our crystallographic observations can be supported by experimental data indicating that the two ΔN-ChoK isoforms have different catalytic capabilities. The in vitro activity of ChoK was tested in the presence of HC-3 using ADP as in the crystallization conditions. After incubation, the reaction components were separated with reversed-phase HPLC and analyzed by mass spectrometry. In a preliminary experiment using ΔN-ChoKβ, we observed that the molecular mass of the major peak of native HC-3 was increased by 80 Da, corresponding to the molecular mass of a phosphate group (Fig. 5A), strongly supporting the idea that HC-3 is phosphorylated by the enzyme. In contrast, HC-3 was not phosphorylated by ΔN-ChoKα1 in the same assay (data not shown). Additionally, separation and quantification of ATP, ADP, and AMP by HPLC were done to analyze products after enzymatic reaction and rule out other possibilities such as ATP contamination. When only ADP was incubated with ΔN-ChoKβ, and reaction products were analyzed using HPLC, no ATP product was detected even after 20 h of incubation (supplemental Fig. S3). This indicates that there is no detectable enzymatic activity converting two ADP molecules to ATP and AMP in a manner similar to the reaction catalyzed by adenylate kinases.


Crystal structures of human choline kinase isoforms in complex with hemicholinium-3: single amino acid near the active site influences inhibitor sensitivity.

Hong BS, Allali-Hassani A, Tempel W, Finerty PJ, Mackenzie F, Dimov S, Vedadi M, Park HW - J. Biol. Chem. (2010)

HC-3 phosphorylation by ChoK. A, mass spectra of the supernatant from a reaction mixture of ΔN-ChoKβ in the presence of ADP. The supernatant was incubated for 15 min and contains the major peak at m/z 413.2, corresponding to native HC-3 (top), whereas the spectrum from the same reaction mixture incubated for 5 h reveals the presence of another peak at m/z 493.2 corresponding to Pho-HC-3 (bottom). B, HC-3 phosphorylation activity by ChoK isoforms. The experiments were performed using full-length wild-type ChoKα1 (○), ChoKα2 (▾), and ChoKβ (·) in the presence of [γ-32P]ATP. Data shown represent the means of triplicate determinations, and error bars indicate the standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871500&req=5

Figure 5: HC-3 phosphorylation by ChoK. A, mass spectra of the supernatant from a reaction mixture of ΔN-ChoKβ in the presence of ADP. The supernatant was incubated for 15 min and contains the major peak at m/z 413.2, corresponding to native HC-3 (top), whereas the spectrum from the same reaction mixture incubated for 5 h reveals the presence of another peak at m/z 493.2 corresponding to Pho-HC-3 (bottom). B, HC-3 phosphorylation activity by ChoK isoforms. The experiments were performed using full-length wild-type ChoKα1 (○), ChoKα2 (▾), and ChoKβ (·) in the presence of [γ-32P]ATP. Data shown represent the means of triplicate determinations, and error bars indicate the standard deviations.
Mentions: Given the possibility that ΔN-ChoKβ is capable of phosphorylating HC-3, additional experiments were performed to verify whether our crystallographic observations can be supported by experimental data indicating that the two ΔN-ChoK isoforms have different catalytic capabilities. The in vitro activity of ChoK was tested in the presence of HC-3 using ADP as in the crystallization conditions. After incubation, the reaction components were separated with reversed-phase HPLC and analyzed by mass spectrometry. In a preliminary experiment using ΔN-ChoKβ, we observed that the molecular mass of the major peak of native HC-3 was increased by 80 Da, corresponding to the molecular mass of a phosphate group (Fig. 5A), strongly supporting the idea that HC-3 is phosphorylated by the enzyme. In contrast, HC-3 was not phosphorylated by ΔN-ChoKα1 in the same assay (data not shown). Additionally, separation and quantification of ATP, ADP, and AMP by HPLC were done to analyze products after enzymatic reaction and rule out other possibilities such as ATP contamination. When only ADP was incubated with ΔN-ChoKβ, and reaction products were analyzed using HPLC, no ATP product was detected even after 20 h of incubation (supplemental Fig. S3). This indicates that there is no detectable enzymatic activity converting two ADP molecules to ATP and AMP in a manner similar to the reaction catalyzed by adenylate kinases.

Bottom Line: This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays.Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta.Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKalpha (alpha1 and alpha2) and ChoKbeta isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKalpha1 and ChoKbeta isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKalpha1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKbeta was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

Show MeSH
Related in: MedlinePlus