188 Nucleic Acids Research, 2007, Vol. 35, No. 1 eluted by the same washing buffer containing 200 mM imidazole. After protein concentration by centrifugation, the protein was injected onto a S-200 gel ﬁltration column (GE Healthcare) equilibrated with 20 mM HEPES (pH 7.5), 200 mM sodium citrate and 2 mM EDTA. The fractions containing the enzyme were concentrated to 20 mg/ml and stored at 80 C. Table 1. Data collection and refinement statistics PDB ID Beamline Wavelength (A˚ ) Temperature (K) Resolution (A˚ ) Reflections Observed Unique Completeness (%) Rsym(%) I/s(I) Space group Unit cell (A˚ ) a b c Molecules per a.u. Refinement statistics Rcryst (%) Rfree (%) Resolution range (A˚ ) Number of atoms Protein Nucleoside ADP Water R.m.s. deviation Bond length (A˚ ) Bond angles ( ) Average B-factors (A˚ 2) Protein Main chain Side chain ADP Nucleoside Waters Human C4S-dCK 3TC-ADP 2NOA SERCAT BM-22 1.0 100 1.8 (1.8–1.9) 156 829 50 033 95.3 (74.8) 5.9 (43.5) 12.0 (2.3) C2221 52.6 134.2 154.9 2 19.2 23.3 30–1.8 2014 (chain A) 1842 (chain B) 15 · 2 27 · 2 197 0.015 1.665 26 (A) 28 (B) 25 (A) 27 (B) 27 (A) 29 (B) 19 (A) 21 (B) 20 (A) 24 (B) 32 TRO-ADP 2NO9 SERCAT BM-22 1.0 100 2.15 (2.15–2.20) 156 130 30 536 99.7 (100.0) 9.9 (51.1) 10.9 (4.4) C2221 52.9 134.5 155.3 2 19.5 25.5 30–2.15 1978 (A) 1872 (B) 15 · 2 27 · 2 79 0.016 1.718 36 (A) 38 (B) 35 (A) 37 (B) 37 (A) 39 (B) 29 (A) 28 (B) 28 (A) 32 (B) 38 Ternary complexes were formed by mixing each nucleo- side analog (3TC, obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS; TRO, provided by SGX Pharmaceuticals, Inc.) with adenosine diphosphate (ADP) (ﬁnal concentrations 5 mM each) and enzyme (10–20 mg/ml) in 5 mM MgCl2. Crystals were obtai- ned under identical conditions as for wild-type dCK (17) via hanging drop vapor diffusion against a reservoir containing 0.95–1.5 M sodium citrate and 100 mM HEPES (pH 7.5). Data collection and processing X-ray data were obtained under standard cryogenic condi- tions at the Advanced Photon Source using SERCAT beamline BM-22. Diffraction data were indexed, scaled and merged using XDS and XSCALE (18) (see Table 1 for data collection statistics). Structure determination and refinement Ternary complex structures were determined via molecular replacement [MOLREP (19)] using the structure of wild- type dCK-D-dC/ADP (17) [PDB Code: 1P60 (20)] as the search model. Reﬁnement was carried out using REFMAC (21) and simulated annealing maps were calculated using CNS (22) (see Table 1 for reﬁnement statistics). Steady-state kinetic assay A spectroscopic enzyme-coupled assay was used to determine enzyme activity as described previously (23). This assay cou- ples the production of ADP or UDP by dCK to the phospho- rylation of phosphoenolpyruvate (PEP) by pyruvate kinase, followed by oxidation of NADH by lactate dehydrogenase. The decrease in NADH absorption is proportional to the ADP/UDP produced by dCK. The extinction coefﬁcient of NADH limits the precise measurement of Km values below 3 mM. For this reason, substrates displaying a Km value below 3 mM are reported in Table 2 to this higher limit. The measurement of kcat values is unaffected by this technical limitation of the assay. Experiments were conducted at 37 C in a kinetic buffer containing 50 mM Tris–HCl (pH 7.5), 100 mM KCl and 5 mM MgCl2 with dCK: 0.35 mM, adeno- sine triphosphate (ATP)-Mg: 1 mM and uridine triphosphate (UTP)-Mg: 1 mM. Nucleoside/nucleoside analog concentra- tions were varied between 5 and 100 mM. Validation of choice of crystallization protein To verify that the Cys!Ser mutations in C4S-dCK did not perturb the structure, ternary complex crystals of the C4S kcat/Km (ATP) s 1M 1 kcat/Km (UTP) s 1M 1 Table 2. Steady state kinetic data of WT and C4S-dCKa Enzyme-substrate Km (ATP) mM Km (UTP) mM WT-TRO C4S-TRO WT-3TC C4S-3TC WT-L-dC C4S-L-dC WT-D-dC C4S-D-Dc 13.2 ± 0.8 56.8 ± 3.0 3.4 ± 1.0 8.4 ± 1.4 <3 <3 <3 3.0 ± 0.2 10.6 ± 1.3 57.8 ± 7.9 8.0 ± 1.0 14.9 ± 1.3 <3 <3 <3 <3 aValues shown are the averages of at least two experiments and SDs are shown. kcat (ATP) s 1 0.095 ± 0.002 0.192 ± 0.004 0.030 ± 0.001 0.028 ± 0.002 0.012 ± 0.001 0.036 ± 0.001 0.033 ± 0.001 0.173 ± 0.002 kcat (UTP) s 1 0.179 ± 0.006 0.524 ± 0.030 0.102 ± 0.001 0.072 ± 0.002 0.010 ± 0.001 0.042 ± 0.001 0.049 ± 0.001 0.217 ± 0.005 7.2 · 103 3.4 · 103 8.8 · 103 3.3 · 103 >4 · 103 >12 · 103 >11 · 103 57.7 · 103 16.9 · 103 9.1 · 103 12.8 · 103 4.8 · 103 >3 · 103 >14 · 103 >16 · 103 >72 · 103