variant were obtained in the presence of D-dC/ADP (PDB ID 2NO1) and gemcitabine/ADP (PDB ID 2NO0) and compared to structures previously determined with wild-type enzyme (17). These control complexes crystallized in the same space group as the L-nucleoside complexes, and diffracted to 1.9 (D-dC/ADP complex) and 1.8 s (gemcitabine/ADP) resolution (E. Sabini, S. Hazra, M. Konrad and A. Lavie, unpublished data). No signiﬁcant structural differences were noted between corresponding polypeptide chain structures [root mean square deviation (r.m.s.d.) between 241 a carbon atomic positions: wild-type dCK-D-dC/ADP versus C4S- dCK-D-dC/ADP ¼ 0.16 s; dCK-gemcitabine/ADP versus C4S-dCK-gemcitabine/ADP ¼ 0.60 s] or substrate positions. Finally, no signiﬁcant differences in catalytic efﬁciency were observed between wild-type and C4S-dCK for two distinct phosphate donors and multiple acceptors (Table 2). Together, these control experiments validate the use of the C4S mutant for structure determination. When referring to structures of dCK herein, we make no distinction between wild type and the C4S mutant, unless otherwise stated. RESULTS AND DISCUSSION The ternary complex structures of dCK-3TC/ADP and dCK- TRO/ADP were determined at 1.8 and 2.15 s resolution, respectively. Excellent difference electron density maps were observed for the L-nucleoside analogs (Figure 1B and C). After several rounds of model building and reﬁnement, in which the nucleotide ADP and the respective L-nucleoside analogs were modeled into the density, and addition of water molecules, the ﬁnal crystallographic R-factors (Rfrees) con- verged to 19.2% (23.3%) and 19.5% (25.5%) for the 3TC and TRO complexes, respectively (Table 1). When compared to the ternary complex structures of dCK- D-dC/ADP and dCK-L-dC/ADP (E. Sabini, S. Hazra, M. Konrad and A. Lavie, unpublished data), binding of these L-nucleoside analogs does not affect the structure of the polypeptide chain r.m.s.d. for 239 common a-carbon atoms among pairs of ternary complex structures ¼ 0.18–0.30 s] nor the positions of the ADP molecules (Figure 2A). In contrast, the mode of L-nucleoside/L-nucleoside analog binding differs from that of D-nucleosides (Figure 2B and C). The active site environment surrounding each L-nucleoside analog is shown in Figure 3A, accompanied by similar views for dCK-D-dC/ADP and dCK-L-dC/ADP (Figure 3B). D-dC is the mirror image of L-dC (chirality being determined by the location of the CH2OH group at the C40 chiral centre or ribose 40 position, Figures 1A and 3B). 3TC and TRO closely resemble one another, and only differ from L-dC at the 30 position (Figure 1A). All three L-nucleosides bind similarly to dCK. Detailed comparison of the protein–ligand interac- tions, presented schematically in Figure 4, reveals the struc- tural basis for non-enantioselective phosphorylation by dCK. For both 3TC and TRO, the enzyme satisﬁes the same requirements for substrate recognition/catalysis that govern phosphorylation of its primary physiologic substrate, D-dC. First, hydrogen bonding interactions between the pro- tein and the ligand permit the protein to detect the presence of a hydrogen bond donor (NH2) and two hydrogen bond acceptors (N; O) characteristic of the cytosine base Nucleic Acids Research, 2007, Vol. 35, No. 1 189 Figure 2. Structures of dCK-3TC/TRO complexes. (A) Shown are four superimposed structures of human dCK: the C4S variant in complex with 3TC/ADP (yellow), TRO/ADP (magenta) and D-dC/ADP (blue); the WT in complex with D-dC/ADP (gray; PDB ID 1P5Z). The structures in complex with D-dC/ADP of the dCK C4S variant and the WT dCK are basically identical, apart from one loop involved in crystal contact. This validates the use of the C4S mutant as a representative of WT dCK. Binding of L-nucleosides also does not elicit major structural perturbations. Non-carbon atom color coding: oxygen-red, nitrogen-blue, phosphorous-green and sulfur- orange. The N- and C-terminal residues visible in the experimental electron density map are labeled N20 and C260, respectively. (B) Stereo representation of the active site showing hydrogen bonding interactions (some water molecules omitted, for clarity). Curved arrow signifies the c torsion angle around the glycosidic bond. (C) Stereo representation of the active site in an orientation rotated 90 relative to (B) depicting the cytosine base in the aromatic slot with Leu82 and Ile30 sandwiching the sugar. Structures have been deposited in the PDB with accession ID 2NOA for C4S-3TC/ADP, 2NO9 for C4S-TRO/ADP and 2NO1 for C4S-D-dC/ADP.