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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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Related in: MedlinePlus

Structure-based sequence alignment of human ChoK isoforms. The secondary structure elements of ChoKα1 (NCBI accession number NP_001268, PDB code 3G15) and ChoKβ (NCBI accession number NP_005189, PDB code 3FEG) are placed on the top and the bottom of the alignment, respectively. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. In ChoKα2 (NCBI accession number NP_997634), the coding sequences that are missed due to alternative splicing are indicated by black squares at the bottom. The blue triangles indicate the hydrophobic residues forming van der Waals interactions with HC-3. The blue circle indicates the residue (Leu-419 of ChoKα1 and Phe-352 of ChoKβ) affecting the flexibility of the conserved tryptophan residue (Trp-420 of ChoKα1 and Trp-353 of ChoKβ). The red triangle indicates the catalytic base (Asp-306 of ChoKα1 and Asp-242 of ChoKβ) for ATP hydrolysis. The catalytically important regions suggested by Malito et al. (9) are boxed and labeled in cyan (a, ATP-binding loop; b, Brenner's motif; c, choline kinase motif). The alignment was generated with ClustalW (43) and was printed using the ESPript 2.1 software package (44).
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Figure 1: Structure-based sequence alignment of human ChoK isoforms. The secondary structure elements of ChoKα1 (NCBI accession number NP_001268, PDB code 3G15) and ChoKβ (NCBI accession number NP_005189, PDB code 3FEG) are placed on the top and the bottom of the alignment, respectively. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. In ChoKα2 (NCBI accession number NP_997634), the coding sequences that are missed due to alternative splicing are indicated by black squares at the bottom. The blue triangles indicate the hydrophobic residues forming van der Waals interactions with HC-3. The blue circle indicates the residue (Leu-419 of ChoKα1 and Phe-352 of ChoKβ) affecting the flexibility of the conserved tryptophan residue (Trp-420 of ChoKα1 and Trp-353 of ChoKβ). The red triangle indicates the catalytic base (Asp-306 of ChoKα1 and Asp-242 of ChoKβ) for ATP hydrolysis. The catalytically important regions suggested by Malito et al. (9) are boxed and labeled in cyan (a, ATP-binding loop; b, Brenner's motif; c, choline kinase motif). The alignment was generated with ClustalW (43) and was printed using the ESPript 2.1 software package (44).

Mentions: The enzyme choline kinase (ChoK,4 EC 2.7.1.32) catalyzes the Mg·ATP-dependent phosphorylation of choline as the first step in the Kennedy (CDP-choline) pathway, in which choline is incorporated into phosphatidylcholine (1–3). In this reaction, choline is first converted into phosphocholine (Pho-Cho), which then reacts with CTP to form CDP-choline. The Pho-Cho moiety is then transferred to diacylglycerol to produce phosphatidylcholine. This pathway is a major source of phosphatidylcholine, which is a highly abundant class of phospholipids in mammalian cellular membranes and serum (2, 4). Mammalian ChoK exists as three isoforms, encoded by two separate genes (5, 6). In humans, ChoKα1 (457 amino acids) and ChoKα2 (439 amino acids) are derived from a single gene (chk-α) by alternative splicing, while ChoKβ (395 amino acids) is the product of a distinct gene (chk-β). The amino acid sequence identity is ∼56% between ChoKα and ChoKβ (Fig. 1), and both chk-α and chk-β mRNAs, as well as their encoded protein isoforms, are ubiquitously expressed in diverse tissues (7). Each isoform is present as either dimers (homo- or hetero-) or as tetramers in solution and is not active in monomeric form (3), suggesting that, for higher eukaryotes, dimeric ChoK is the minimum functional form. Recently, the crystal structures of ChoK proteins from Caenorhabditis elegans and human have been determined, in which two monomers were dimerized in each asymmetric unit (8, 9).


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)

Structure-based sequence alignment of human ChoK isoforms. The secondary structure elements of ChoKα1 (NCBI accession number NP_001268, PDB code 3G15) and ChoKβ (NCBI accession number NP_005189, PDB code 3FEG) are placed on the top and the bottom of the alignment, respectively. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. In ChoKα2 (NCBI accession number NP_997634), the coding sequences that are missed due to alternative splicing are indicated by black squares at the bottom. The blue triangles indicate the hydrophobic residues forming van der Waals interactions with HC-3. The blue circle indicates the residue (Leu-419 of ChoKα1 and Phe-352 of ChoKβ) affecting the flexibility of the conserved tryptophan residue (Trp-420 of ChoKα1 and Trp-353 of ChoKβ). The red triangle indicates the catalytic base (Asp-306 of ChoKα1 and Asp-242 of ChoKβ) for ATP hydrolysis. The catalytically important regions suggested by Malito et al. (9) are boxed and labeled in cyan (a, ATP-binding loop; b, Brenner's motif; c, choline kinase motif). The alignment was generated with ClustalW (43) and was printed using the ESPript 2.1 software package (44).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structure-based sequence alignment of human ChoK isoforms. The secondary structure elements of ChoKα1 (NCBI accession number NP_001268, PDB code 3G15) and ChoKβ (NCBI accession number NP_005189, PDB code 3FEG) are placed on the top and the bottom of the alignment, respectively. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. In ChoKα2 (NCBI accession number NP_997634), the coding sequences that are missed due to alternative splicing are indicated by black squares at the bottom. The blue triangles indicate the hydrophobic residues forming van der Waals interactions with HC-3. The blue circle indicates the residue (Leu-419 of ChoKα1 and Phe-352 of ChoKβ) affecting the flexibility of the conserved tryptophan residue (Trp-420 of ChoKα1 and Trp-353 of ChoKβ). The red triangle indicates the catalytic base (Asp-306 of ChoKα1 and Asp-242 of ChoKβ) for ATP hydrolysis. The catalytically important regions suggested by Malito et al. (9) are boxed and labeled in cyan (a, ATP-binding loop; b, Brenner's motif; c, choline kinase motif). The alignment was generated with ClustalW (43) and was printed using the ESPript 2.1 software package (44).
Mentions: The enzyme choline kinase (ChoK,4 EC 2.7.1.32) catalyzes the Mg·ATP-dependent phosphorylation of choline as the first step in the Kennedy (CDP-choline) pathway, in which choline is incorporated into phosphatidylcholine (1–3). In this reaction, choline is first converted into phosphocholine (Pho-Cho), which then reacts with CTP to form CDP-choline. The Pho-Cho moiety is then transferred to diacylglycerol to produce phosphatidylcholine. This pathway is a major source of phosphatidylcholine, which is a highly abundant class of phospholipids in mammalian cellular membranes and serum (2, 4). Mammalian ChoK exists as three isoforms, encoded by two separate genes (5, 6). In humans, ChoKα1 (457 amino acids) and ChoKα2 (439 amino acids) are derived from a single gene (chk-α) by alternative splicing, while ChoKβ (395 amino acids) is the product of a distinct gene (chk-β). The amino acid sequence identity is ∼56% between ChoKα and ChoKβ (Fig. 1), and both chk-α and chk-β mRNAs, as well as their encoded protein isoforms, are ubiquitously expressed in diverse tissues (7). Each isoform is present as either dimers (homo- or hetero-) or as tetramers in solution and is not active in monomeric form (3), suggesting that, for higher eukaryotes, dimeric ChoK is the minimum functional form. Recently, the crystal structures of ChoK proteins from Caenorhabditis elegans and human have been determined, in which two monomers were dimerized in each asymmetric unit (8, 9).

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