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

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.

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).

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.

FIGURE 3.

Overall structures of ΔN-ChoK ternary complexes. A, ribbon diagrams of the crystal structures of ΔN-ChoKα1 (pink) and β (lime green) ternary complexes. Adenine nucleotides, HC-3, and Pho-HC-3 are shown within the sigma-weighted F_o − F_c omit map (blue) contoured at the 2.5 σ level and indicated by arrows. One monomer in the dimeric ΔN-ChoK models is indicated. B, close-up stereo view of the HC-3-binding site in the ΔN-ChoKα1·ADP·HC-3 complex structure. The difference Fourier map for HC-3 is drawn at a contour level of 2.5 σ. The HC-3 (yellow) and its surrounding hydrophobic residues (pink) are marked and represented in stick mode. C, close-up stereo view of the HC-3-binding site in the ΔN-ChoKβ·ADP·Pho-HC-3 complex structure. The difference Fourier map corresponding to Pho-HC-3 is drawn at a contour level of 2.5 σ. The Pho-HC-3 (yellow) and its surrounding hydrophobic residues (lime green) are marked and shown in stick mode. The inset displays the phosphorylated region of Pho-HC-3 at a slightly different angle. All of the figures were generated using PyMOL (DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific LLC, San Carlos, CA).

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.

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.

FIGURE 6.

Structural comparison around the HC-3-binding groove. A, superposition of the crystal structures of both ΔN-ChoK ternary complexes. Ribbon diagrams for each structure are represented in the same colors as in Fig. 3. The disordered region, including 18 residues that are only present in ChoKα1, is schematically shown as a pink dotted line. HC-3 and Pho-HC-3 are colored magenta and green, respectively. The inset shows an enlarged view around the inhibitor-binding grooves of superimposed ternary complexes. The Lα9α10 region is indicated, and the residues that form hydrophobic contacts with HC-3 are shown in stick representation and labeled by the sequence number of ChoKα1. Two residues (Ile-366 and Glu-367) in ΔN-ChoKβ model that are excluded from the interaction with HC-3 due to a flipped-out conformation of Lα9α10 are indicated in parenthesis by the sequence number of ChoKβ. B, a close-up view of the interaction between Leu-419 and Trp-420 in the ΔN-ChoKα1·ADP·HC-3 complex structure. The side chains of these residues are arranged parallel to each other. C, a close-up view of the interaction between Phe-352 and Trp-353 in the ΔN-ChoKβ·ADP·Pho-HC-3 complex structure. These two side chains are oriented perpendicular to each other. D, a close-up view of superimposed B and C. The dotted arrow indicates the possible conformational change of the Trp-420 side chain upon HC-3 binding inside the choline-binding pocket of ΔN-ChoKα1.