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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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Chemical structures of HC-3 (A) and choline (B). Specific moieties of HC-3 are indicated at the top of the molecule. C, schematic illustration of ΔN-ChoKβ interactions with Pho-HC-3. Pho-HC-3 is represented in ball-and-stick and is colored purple. Residues forming van der Waals' interactions are indicated by an arc with radiating spokes toward the ligand atom they contact; those residues participating in hydrogen bonding are colored in lime green and shown in ball-and-stick representation. Hydrogen bonds are indicated as red dotted lines with distances in angstroms. Carbon atoms are colored in black, oxygen atoms are in red, nitrogen atoms are in blue, and phosphorus is colored in orange. The figure was generated with the program LIGPLOT (47) followed by manual editing.
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Figure 2: Chemical structures of HC-3 (A) and choline (B). Specific moieties of HC-3 are indicated at the top of the molecule. C, schematic illustration of ΔN-ChoKβ interactions with Pho-HC-3. Pho-HC-3 is represented in ball-and-stick and is colored purple. Residues forming van der Waals' interactions are indicated by an arc with radiating spokes toward the ligand atom they contact; those residues participating in hydrogen bonding are colored in lime green and shown in ball-and-stick representation. Hydrogen bonds are indicated as red dotted lines with distances in angstroms. Carbon atoms are colored in black, oxygen atoms are in red, nitrogen atoms are in blue, and phosphorus is colored in orange. The figure was generated with the program LIGPLOT (47) followed by manual editing.

Mentions: In an effort to develop new anti-cancer therapies, numerous compounds have been synthesized and tested as ChoK inhibitors (20, 25–27). Most of these compounds are derivatives of hemicholinium-3 (HC-3), a known competitive inhibitor of ChoK with a structural homology to choline (Fig. 2, A and B) (28). Although several HC-3-derived inhibitors are under investigation for their potential clinical applicability, their efficacy is still uncertain. Most reported ChoK inhibitors, including HC-3, contain long or bulky hydrophobic aromatic chains, whereas the choline-binding pocket has an overall negative electrostatic potential (8, 9). This reinforces interest in the interaction between ChoK and potential inhibitors, but no detailed structural information has been available so far.


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)

Chemical structures of HC-3 (A) and choline (B). Specific moieties of HC-3 are indicated at the top of the molecule. C, schematic illustration of ΔN-ChoKβ interactions with Pho-HC-3. Pho-HC-3 is represented in ball-and-stick and is colored purple. Residues forming van der Waals' interactions are indicated by an arc with radiating spokes toward the ligand atom they contact; those residues participating in hydrogen bonding are colored in lime green and shown in ball-and-stick representation. Hydrogen bonds are indicated as red dotted lines with distances in angstroms. Carbon atoms are colored in black, oxygen atoms are in red, nitrogen atoms are in blue, and phosphorus is colored in orange. The figure was generated with the program LIGPLOT (47) followed by manual editing.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Chemical structures of HC-3 (A) and choline (B). Specific moieties of HC-3 are indicated at the top of the molecule. C, schematic illustration of ΔN-ChoKβ interactions with Pho-HC-3. Pho-HC-3 is represented in ball-and-stick and is colored purple. Residues forming van der Waals' interactions are indicated by an arc with radiating spokes toward the ligand atom they contact; those residues participating in hydrogen bonding are colored in lime green and shown in ball-and-stick representation. Hydrogen bonds are indicated as red dotted lines with distances in angstroms. Carbon atoms are colored in black, oxygen atoms are in red, nitrogen atoms are in blue, and phosphorus is colored in orange. The figure was generated with the program LIGPLOT (47) followed by manual editing.
Mentions: In an effort to develop new anti-cancer therapies, numerous compounds have been synthesized and tested as ChoK inhibitors (20, 25–27). Most of these compounds are derivatives of hemicholinium-3 (HC-3), a known competitive inhibitor of ChoK with a structural homology to choline (Fig. 2, A and B) (28). Although several HC-3-derived inhibitors are under investigation for their potential clinical applicability, their efficacy is still uncertain. Most reported ChoK inhibitors, including HC-3, contain long or bulky hydrophobic aromatic chains, whereas the choline-binding pocket has an overall negative electrostatic potential (8, 9). This reinforces interest in the interaction between ChoK and potential inhibitors, but no detailed structural information has been available so far.

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