190 Nucleic Acids Research, 2007, Vol. 35, No. 1 (Figure 2B). Second, the ﬁve-membered ring resembling the ribose is positioned so as to place the site of phosphorylation (50-OH) in the appropriate location for phosphoryl transfer from ATP. Figure 3. Interactions activating the 50-OH of the nucleosides for nucleophilic attack. (A) Stereoview depicting the interactions made by the 50-OH groups of 3TC (yellow) and TRO (magenta). (B) Stereoview depicting the analogous interactions for D-dC (blue) and L-dC (cyan). Despite the difference in chirality between the physiologic substrate D-dC and the L-nucleoside analogs 3TC/TRO, the location of the cytosine base and its interactions with the enzyme are comparable. In all three ternary complex struc- tures depicted in Figure 2B, Asp133 forms hydrogen bonds with the cytosine amino group. However, it is Gln97 that serves as the primary cytosine recognition element contribut- ing two hydrogen bonds between its side chain and the com- ponents of the base that normally engage in Watson–Crick type hydrogen bonding with guanosine (Gln97 O!NH2; Gln97 NH2!N; Figures 2B and 4A). The fourth hydrogen bonding interaction between the protein and the base appears as a water bridge (Tyr204/Tyr86!H2O!O; Figures 2B and 4A). Analogous interactions with comparable hydrogen bond geometries occur between dCK and the cytosine bases of D-dC and L-dC (data not shown). The base itself sits deep in an aromatic slot, which is lined by the side chains of Phe137, Phe96 and Trp58 (all conserved among dCK, dGK and TK2). The aromatic cytosine base is pinioned between Phe137 and Phe96/Trp58. On one face, Phe137 engages in p–p stacking against the cytosine base (interplanar distance 3.5 s). On the opposite face, Phe96 and Trp58 both engage in edge-to-face interactions that place electropositive aro- matic hydrogen atoms close to the electronegative p-electron cloud of the cytosine base (Figures 2C and 4B). These aromatic–aromatic interactions exploit enthalpically favorable quadrupole–quadrupole interactions characteristic of aromatic amino acid side chains in proteins as also seen in crystalline benzene (24). The important systematic difference in the posi- tion of the cytosine bases of 3TC and TRO versus that of D-dC is a 10 tilt (Figure 2C). We believe that this difference is a general feature of L-enantiomers, as the same base tilt is repro- duced in the structure of L-dC bound to dCK (E. Sabini, S. Hazra, M. Konrad and A. Lavie, unpublished data). Figure 4. Enzyme-substrate interactions. Schematic representation of the (A) polar and (B) weakly polar and hydrophobic interactions made by 3TC and TRO with the residues in the dCK active site. Distances (A˚ ) are color-coded in (A) with 3TC-yellow and TRO-magenta, while in (B) they are in black and correspond to the average closest contacts for both structures. Water molecules are represented as cyan balls.