Structure of human uridine-cytidine kinase 2 determined by SIRAS using a rotating-anode X-ray generator and a single samarium derivative

Abstract

Uridine-cytidine nucleoside kinase 2 (UCK2) is the ratelimiting enzyme in the pyrimidine-nucleotide salvage pathway. UCK2 catalyzes the phosphorylation of the natural ribonucleosides cytidine and uridine to cytidine 5 H -monophosphate (CMP) and uridine 5 H -monophosphate (UMP), respectively, and activates several important frontline antimetabolite drugs. The present contribution reports the rapid crystal structure determination of human UCK2 complexed with a magnesium ion and the reaction products adenosine 5 H -diphosphate (ADP) and CMP. Diffraction data were collected on a copper rotating-anode X-ray generator from one native UCK2 crystal and a single samarium-derivative crystal. Utilizing the relatively high anomalous signal from the samarium derivative at the Cu K wavelength, the structure was determined by single isomorphous replacement and single anomalous signal (SIRAS) phasing techniques. Two of the four major samarium sites are located in the active sites of the two UCK2 molecules that form the asymmetric unit and appear to displace the magnesium ions present in the native crystals. The crystal structures of UCK2 alone and in complex with various ligands have recently been determined using traditional multiple isomorphous replacement (MIR) phasing techniques and data from three heavy-atom derivatives. The reported structures validate our independently determined structure. Of more than 1000 kinase crystal structure entries in the Protein Data Bank, less than 1% of them have been determined by SIRAS. For the published kinase crystal structures determined by SIRAS, all data were reportedly collected at various synchrotron-radiation facilities. This study demonstrates that diffraction data collected from a single samarium derivative using Cu K radiation provides suf®cient phasing power to determine a novel macromolecular crystal structure. PDB Reference: human uridine-cytidine kinase 2, 1xrj, r1xrjsf.

Uridine‐cytidine kinase 2 (UCK2): A potential diagnostic and prognostic biomarker for lung cancer

“…Uridine‐cytidine kinase 2 is an enzyme encoded by the UCK2 gene located on chromosome 1q22‐23.2 and 1 of 2 human UCKs . The other UCK protein, UCK1, has 70% sequence homology with UCK2 . Both of them can catalyze the phosphorylation of uridine and cytidine to the monophosphate form, which plays a key role in the biosynthesis of the pyrimidine nucleotides that constitute DNA and RNA .…”

In-house SAD phasing with surface-bound cerium ions

“…From the perspective of in-house SAD phasing, sulfur SAD and halide soaking have been successfully utilized for various proteins Dauter et al, 2000). Other heavy atoms used for derivatization and for SAD phasing include Hg, Ba (Moiseeva & Allaire, 2007), Sm (Appleby et al, 2005), Rb (Korolev et al, 2001), Cs, Gd (Nagem et al, 2001), Cd (Yogavel et al, 2010), Ho (Ismaya et al, 2011;Jakoncic et al, 2006) and Co (Gunčar et al, 2007). In addition to monovalent, divalent and trivalent cations and anions, compounds that contain the heavy atom as one of their constituents, such as I3C (Beck et al, 2008) and the so-called 'magic seven' compounds including KAuCl 4 , K 2 PtCl 6 and tetramethyllead acetate (Sun et al, 2002), have also been reported to be successful in SAD phasing.…”

In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica

“…Phosphorylation of UMP and CMP are an essential requirement to form 5′-triphosphate nucleotides required for gene synthesis. UCK2 have been reported to be expressed only in the placenta and its over expression have been implicated in several rapidly proliferating cancer cells [6,7]. Therefore, the selective expression of UCK2 in cancer cells makes it a potential target for cancer chemotherapy.…”