Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 SINGLE AMINO ACID NEAR THE ACTIVE SITE INFLUENCES INHIBITOR SENSITIVITY

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK alpha (alpha 1 and alpha 2) and ChoK beta 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 ChoK alpha 1 and ChoK beta isoforms in complex with hemi-cholinium-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 ChoK alpha 1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK beta 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 ChoK alpha isoforms (alpha 1 and alpha 2) compared with ChoK beta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK alpha 2 (equivalent to Leu-419 of ChoK alpha 1), or the corresponding residue Phe-352 of ChoK beta, 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.