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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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Close-up stereo view of HC-3 and Pho-Cho superimposed on the choline-binding pocket. The structure of the ChoKα·Pho-Cho complex was (PDB code 2CKQ) overlaid on the crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex (pink surface), and the ligands shown in the comparison reflect only the protein superposition. Both Pho-Cho and HC-3 are represented in stick mode and are colored cyan and yellow, respectively. Some of the key residues forming the choline-binding pocket are indicated, and the hydrophilic residues, including a catalytic base (Asp-306), and the hydrophobic residues are shown in green and brown surfaces, respectively.
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Figure 4: Close-up stereo view of HC-3 and Pho-Cho superimposed on the choline-binding pocket. The structure of the ChoKα·Pho-Cho complex was (PDB code 2CKQ) overlaid on the crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex (pink surface), and the ligands shown in the comparison reflect only the protein superposition. Both Pho-Cho and HC-3 are represented in stick mode and are colored cyan and yellow, respectively. Some of the key residues forming the choline-binding pocket are indicated, and the hydrophilic residues, including a catalytic base (Asp-306), and the hydrophobic residues are shown in green and brown surfaces, respectively.

Mentions: The crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex reveals that HC-3 bound to a groove on the C-terminal lobe near the interlobe cleft in a manner where one oxazinium ring occupied the choline-binding pocket, and the other oxazinium ring was partially exposed to solvent (Fig. 3A). The HC-3-binding groove was lined by hydrophobic residues (Tyr-354, Phe-361, Trp-420, Trp-423, Ile-433, Phe-435, Tyr-437, and Tyr-440), and only one side of the planar HC-3 molecule contributed to the hydrophobic interaction with the groove (Fig. 3B). The HC-3 oxazinium ring that occupied the choline-binding pocket could be superimposed onto the choline moiety modeled from the crystal structure of ChoKα in complex with Pho-Cho (PDB code 2CKQ) (Fig. 4), providing direct structural evidence supporting the idea that HC-3 competes with choline for the same binding pocket on ChoK (28).


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)

Close-up stereo view of HC-3 and Pho-Cho superimposed on the choline-binding pocket. The structure of the ChoKα·Pho-Cho complex was (PDB code 2CKQ) overlaid on the crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex (pink surface), and the ligands shown in the comparison reflect only the protein superposition. Both Pho-Cho and HC-3 are represented in stick mode and are colored cyan and yellow, respectively. Some of the key residues forming the choline-binding pocket are indicated, and the hydrophilic residues, including a catalytic base (Asp-306), and the hydrophobic residues are shown in green and brown surfaces, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Close-up stereo view of HC-3 and Pho-Cho superimposed on the choline-binding pocket. The structure of the ChoKα·Pho-Cho complex was (PDB code 2CKQ) overlaid on the crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex (pink surface), and the ligands shown in the comparison reflect only the protein superposition. Both Pho-Cho and HC-3 are represented in stick mode and are colored cyan and yellow, respectively. Some of the key residues forming the choline-binding pocket are indicated, and the hydrophilic residues, including a catalytic base (Asp-306), and the hydrophobic residues are shown in green and brown surfaces, respectively.
Mentions: The crystal structure of the ΔN-ChoKα1·ADP·HC-3 complex reveals that HC-3 bound to a groove on the C-terminal lobe near the interlobe cleft in a manner where one oxazinium ring occupied the choline-binding pocket, and the other oxazinium ring was partially exposed to solvent (Fig. 3A). The HC-3-binding groove was lined by hydrophobic residues (Tyr-354, Phe-361, Trp-420, Trp-423, Ile-433, Phe-435, Tyr-437, and Tyr-440), and only one side of the planar HC-3 molecule contributed to the hydrophobic interaction with the groove (Fig. 3B). The HC-3 oxazinium ring that occupied the choline-binding pocket could be superimposed onto the choline moiety modeled from the crystal structure of ChoKα in complex with Pho-Cho (PDB code 2CKQ) (Fig. 4), providing direct structural evidence supporting the idea that HC-3 competes with choline for the same binding pocket on ChoK (28).

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