Nucleic Acids Research, 2007, Vol. 35, No. 1 187 Figure 1. (A) Schematic of the D- and L-nucleosides (all in b-form): 3TC, ( )-L-20,30-dideoxy-30-thiacytidine (Lamivudine, Epivir ); TRO, ( )-L-20, 30-dideoxy-30-oxacytidine (Troxacitabine, Troxatyl ); D-dC, D-20-deoxycytidine, and L-dC, L-20-deoxycytidine. Experimental electron density map for the L-nucleoside analogs: (B) 3TC is shown in yellow and (C) TRO in magenta. The difference map (|Fobs| |Fcalc|) is contoured at 3s (light blue) and 8s (orange). Note the strong signal given by the sulfur atom in 3TC even at the high sigma cut-off. Figures were generated using Bobscript (26) and Molscript (27), and rendered with Raster3D (28). anti-cancer agent cytarabine (AraC), TRO is not a substrate for the drug-modifying enzyme cytidine deaminase (6) that confers cytarabine resistance in AML (8). Metabolic conversion of 3TC and TRO to pharmacologi- cally active drugs requires serial phosphorylation to their triphosphorylated forms. The essential ﬁrst step in this process is catalyzed by deoxycytidine kinase (dCK) (6,9–11), which together with the two mitochondrial enzymes thymidine kinase (TK2) and deoxyguanosine kinase (dGK), constitute a family of three closely related non-enantioselective enzymes responsible for nucleoside phosphorylation (12,13). Lack of selectivity pertains to both substrates and enantiomers. dCK phosphorylates dC, dA and dG. TK2 phosphorylates both dC and deoxythymidine (dT), and dGK phosphorylates both dA and dG. The overlapping substrate speciﬁcity of these nucleo- side kinases reﬂects their differing sub-cellular localization: dCK is cytosolic, whereas TK2 and dGK are mitochondrial. All three enzymes are capable of phosphorylating both L- and D-enantiomers (14–16). The question that we set out to answer is what structural features endow certain enzymes, such as dCK, the ability to accept substrates with non-physiological chirality? Answer- ing this question is signiﬁcant not only for our understanding of enzyme speciﬁcity, but also has medicinal ramiﬁcations as more L-nucleoside analogs enter clinical trials. To address this issue, we solved the crystal structures of human dCK in complex with the two clinically relevant L-nucleoside analogs, 3TC and TRO. This is the ﬁrst report of an enzyme in complex with an L-nucleoside. MATERIALS AND METHODS Protein crystal preparation Initial attempts to crystallize wild-type dCK with 3TC and TRO were hampered by reproducibility. Modiﬁcation of all four surface-exposed cysteine residues yielded a signiﬁcantly better behaved crystallization candidate, thereby making this study feasible. The dCK variant containing the C9S/ C45S/C59S/C146S mutations, designated C4S-dCK, was cre- ated from the wild-type dCK gene, using QuickChange (Stratagene, Inc.). The protein was produced in Escherichia coli and puriﬁed to homogeneity (E. Sabini, S. Hazra, M. Konrad and A. Lavie, manuscript in preparation) In short, E.coli BL21(DE3) harboring the pET14b expression plasmids were grown in 2YT media at 37 C, induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), and harvested by centrifugation after 4 h. After lysis by sonica- tion and ultracentrifugation, the supernatant was loaded onto a HisTrap HP Ni Sepharose column (GE Healthcare) and washed with buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl and 20 mM imidazole. The enzyme was