Structure of human uridine-cytidine kinase 2 determined by SIRAS using a rotating-anode X-ray generator and a single samarium derivative.
Uridine-cytidine nucleoside kinase 2 (UCK2) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. UCK2 catalyzes the phosphorylation of the natural ribonucleosides cytidine and uridine to cytidine 5'-monophosphate (CMP) and uridine 5'-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'-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 Kalpha 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 Kalpha radiation provides sufficient phasing power to determine a novel macromolecular crystal structure.
Appleby
Todd C
TC
Drug Discovery, Valeant Pharmaceuticals International, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA.
Larson
Gary
G
Cheney
I Wayne
IW
Walker
Heli
H
Wu
Jim Z
JZ
Zhong
Weidong
W
Hong
Zhi
Z
Yao
Nanhua
N
D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D012493:Samarium / Q000737:chemistry*; D014541:Uridine Kinase / Q000737:chemistry*
D012493:Samarium; C483834:UCK2 protein, human; D014541:Uridine Kinase