chirality is a hallmark of biological systems : proteins are composed of l - amino acids and dna and rna are composed of -d - nucleotides .
it has , therefore , been widely accepted that enzymes that must correctly bind ligands and catalyze chemistry on their respective substrates , will invariably show high chiral selectivity .
quite unexpectedly , however , it was demonstrated that it is the l - component of the racemic mixture ( )-bch-189 [ ( )--2,3-dideoxy-3-thiacytidine ] , which possesses significantly better anti - human immunodeficiency virus ( hiv ) properties than its d - enantiomeric counterpart ( 1,2 ) . since this discovery ,
two non - physiologic l - nucleoside analogs [ ( )--2,3-dideoxy-3-thiacytidine ( 3tc ) , the l - enantiomer of bch-189 , and its 5-fluorinated analog ftc ] have been approved for treatment of human acquired immune deficiency syndrome ( aids ) .
an additional six d - nucleoside analogs have been approved as aids therapeutics , and it is customary for patients to receive both l- and d - enantiomer anti - hiv agents simultaneously .
the pharmacological potential of l - nucleoside analogs has been recently reviewed ( 35 ) .
the dioxolane analog of 3tc , troxacitabine [ tro , ( )-l--2,3-dideoxy-3-oxacytidine ; figure 1a ] is currently in a phase ii / iii clinical trial for third - line treatment of acute myelogenous leukemia ( aml ) , a blood cancer .
the l - nucleoside analogs 3tc and ftc are preferred as anti - virals over their d - analogs [ ( + ) -bch-189 and ( + ) -ftc , respectively ] , primarily because these hiv reverse transcriptase inhibitors do not affect cellular dna polymerases ( 4 ) , resulting in minimal toxicity .
in contrast , tro , the first l - nucleoside analog to be used as a cytotoxic anti - cancer agent ( 6 ) , which differs from 3tc by only one atom ( s versus o in the five membered ring , figure 1a ) , has potent anti - viral activity yet displays substantial cellular toxicity due to inhibition of dna polymerases  ,  and  ( 7 ) .
unlike the d - nucleoside analog 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) . ( a ) schematic of the d- and l - nucleosides ( all in -form ) : 3tc , ( )-l-2,3-dideoxy-3-thiacytidine ( lamivudine , epivir ) ; tro , ( )-l-2,3-dideoxy-3-oxacytidine ( troxacitabine , troxatyl ) ; d - dc , d-2-deoxycytidine , and l - dc , l-2-deoxycytidine . experimental electron density map for the l - nucleoside analogs : ( b )
the difference map ( |fobs|  |fcalc| ) is contoured at 3 ( light blue ) and 8 ( 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 ) .
metabolic conversion of 3tc and tro to pharmacologically active drugs requires serial phosphorylation to their triphosphorylated forms .
the essential first step in this process is catalyzed by deoxycytidine kinase ( dck ) ( 6,911 ) , 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 ) .
tk2 phosphorylates both dc and deoxythymidine ( dt ) , and dgk phosphorylates both da and dg .
the overlapping substrate specificity of these nucleoside 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 ( 1416 ) .
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 ? answering 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 .
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 created 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 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication 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 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 80c .
ternary complexes were formed by mixing each nucleoside 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 ( 1020 mg / ml ) in 5 mm mgcl2 .
crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) .
x - ray data were obtained under standard cryogenic conditions 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 ) .
data collection and refinement statistics 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 ) .
a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .
this assay couples the production of adp or udp by dck to the phosphorylation 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 m . for this reason ,
substrates displaying a km value below 3 m are reported in table 2 to this higher limit .
experiments were conducted at 37c in a kinetic buffer containing 50 mm tris  hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .
steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown . to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant
were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 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  ( 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  carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16  ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60  ] 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 .
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 created 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 37c , induced with 0.1 mm isopropyl--d - thiogalactopyranoside ( iptg ) , and harvested by centrifugation after 4 h. after lysis by sonication 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 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 80c .
ternary complexes were formed by mixing each nucleoside 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 ( 1020 mg / ml ) in 5 mm mgcl2 .
crystals were obtained under identical conditions as for wild - type dck ( 17 ) via hanging drop vapor diffusion against a reservoir containing 0.951.5 m sodium citrate and 100 mm hepes ( ph 7.5 ) .
x - ray data were obtained under standard cryogenic conditions 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 ) .
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 ) .
a spectroscopic enzyme - coupled assay was used to determine enzyme activity as described previously ( 23 ) .
this assay couples the production of adp or udp by dck to the phosphorylation 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 m . for this reason ,
substrates displaying a km value below 3 m are reported in table 2 to this higher limit .
experiments were conducted at 37c in a kinetic buffer containing 50 mm tris  hcl ( ph 7.5 ) , 100 mm kcl and 5 mm mgcl2 with dck : 0.35 m , adenosine triphosphate ( atp)-mg : 1 mm and uridine triphosphate ( utp)-mg : 1 mm .
steady state kinetic data of wt and c4s - dck values shown are the averages of at least two experiments and sds are shown .
to verify that the cysser mutations in c4s - dck did not perturb the structure , ternary complex crystals of the c4s variant were obtained in the presence of d - dc / adp ( pdb i d 2no1 ) and gemcitabine / adp ( pdb i d 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  ( 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  carbon atomic positions : wild - type dck - d - dc / adp versus c4s - dck - d - dc / adp = 0.16  ; dck - gemcitabine / adp versus c4s - dck - gemcitabine / adp = 0.60  ] 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 .
the ternary complex structures of dck-3tc / adp and dck - tro / adp were determined at 1.8 and 2.15  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 ) converged 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 -carbon atoms among pairs of ternary complex structures = 0.180.30  ] 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 ) .
( 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 i d 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 .
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 ) .
( 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 i d 2noa for c4s-3tc / adp , 2no9 for c4s - tro / adp and 2no1 for c4s - d - dc / adp . 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 c4 chiral centre or ribose 4 position , figures 1a and 3b ) .
3tc and tro closely resemble one another , and only differ from l - dc at the 3 position ( figure 1a ) .
detailed comparison of the protein  ligand interactions , presented schematically in figure 4 , reveals the structural 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 protein 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 ( figure 2b ) .
second , the five - membered ring resembling the ribose is positioned so as to place the site of phosphorylation ( 5-oh ) in the appropriate location for phosphoryl transfer from atp .
( a ) stereoview depicting the interactions made by the 5-oh groups of 3tc ( yellow ) and tro ( magenta ) .
( b ) stereoview depicting the analogous interactions for d - dc ( blue ) and l - dc ( cyan ) .
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 (  ) 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 . 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 structures 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 contributing two hydrogen bonds between its side chain and the components of the base that normally engage in watson  crick type hydrogen bonding with guanosine ( gln97 onh2 ; gln97 nh2n ; figures 2b and 4a ) .
the fourth hydrogen bonding interaction between the protein and the base appears as a water bridge ( tyr204/tyr86h2oo ; 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 ) .
phe137 engages in  stacking against the cytosine base ( interplanar distance 3.5  ) . on the opposite face , phe96 and trp58 both engage in edge - to - face interactions that place electropositive aromatic hydrogen atoms close to the electronegative -electron cloud of the cytosine base ( figures 2c and 4b ) .
quadrupole interactions characteristic of aromatic amino acid side chains in proteins as also seen in crystalline benzene ( 24 ) .
the important systematic difference in the position 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 reproduced in the structure of l - dc bound to dck ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) .
in contrast to the numerous interactions between the cytosine base and the aromatic slot , the five membered rings of 3tc and tro make only limited enzyme contacts ( figures 3a and 4 ) .
critical for catalysis is correct positioning of the 5-oh , which receives the transferred phosphate group from atp . in the l - nucleoside analog structures depicted in figure 3a , the 5-oh group is hydrogen bonded to the side chain of arg128 .
consequently in each case , the o5-hydroxyl is close to a side chain oxygen atom of glu53 ( 3.13.6  ) , the general base that activates 5-oh group attack of the -phosphate of atp ( see figures 2b and 3 ) . to achieve this critical interaction , concomitant with base tilting , the five - membered rings of 3tc and tro rotate 30 around the glycosidic bond (  , marked with an arrow in figure 2b ) relative to the dck - bound conformation of d - dc .
a comparable  rotation observed in the l - dc structure ( e. sabini , s. hazra , m. konrad and a. lavie , unpublished data ) provides additional evidence that our structural findings represent the way in which l - nucleosides and l - nucleoside analogs bind to dck .
the four structures depicted in figure 3 document that positioning of the 5-oh group in the active site of dck is independent of chirality .
this remarkable property derives from the internal symmetry and conformational flexibility ( i.e. rotation about the glycosidic torsion angle  ) of nucleosides and nucleoside analogs . once the cytosine base is docked into the aromatic slot , the paucity of protein  ligand interactions involving the five - membered ring and the conformational properties of the nucleoside permit appropriate positioning of the 5-oh group . within the aromatic slot ,
possible glycosidic bond torsion angles are limited by the hydrophobic residues ile30 and leu82 ( figures 2c and 4 ) .
an additional degree of freedom dictating the precise location of the hydroxyl is the pucker of the five - membered ring . for 3tc and tro ,
the ring pucker is equivalent to c3 endo , whereas for l - dc and d - dc the sugar pucker is c3 exo .
this difference can be attributed in part to the absence of a 3-oh group on 3tc and on tro , both of which can act as dna chain terminators . finally , a torsion angle rotation about the c4 and c5 bond , from gauche in the d - dc structure to trans in the complex structures of 3tc , tro and l - dc , direct the 5-oh group towards glu53 .
although these features of 3tc and tro conformational flexibility bring the 5-oh group near to glu53 ( 3.13.6  ; figure 3a ) , they do not stabilize the very close , so - called catalytic , interactions seen with l - dc and d - dc ( 2.42.5  ; figure 3b ) .
however , the fact that dck shows similar catalytic efficiency towards 3tc / tro and l - dc ( table 2 ) suggests that the slightly sub - optimal position observed for the 5-oh group in the 3tc / tro structures does not impede catalysis . in conclusion ,
the structures reported herein document that dck phosphorylation of l - nucleosides and l - nucleoside analogs is due to both the nature of the enzyme active site and the nature of the substrates .
we suggest that lack of enantiomeric selectivity may reflect the central role dck plays in nucleotide salvage , i.e. phosphorylating both the pyrimidine dc and the purines da and dg within the cytosolic compartment .
similar arguments can be invoked to explain why the related enzymes dgk , which phosphorylates da and dg , and tk2 , which phosphorylates dt and dc , are not enantioselective .
in contrast , human tk1 , which is restricted to d - dt phosphorylation , is exquisitely enantioselective ( 25 ) .
we suggest that enzymes that evolved to catalyze more than one chemically distinct nucleoside substrate acquired less restrictive active sites , which in turn endowed such enzymes with hitherto unexpected catalytic activities for substrates with non - physiologic chirality .
 l - nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers . 
 these pro - drugs achieve pharmacological activity only after enzyme - catalyzed conversion to their tri - phosphorylated forms . 
 herein , we report the crystal structures of human deoxycytidine kinase ( dck ) in complex with the l - nucleosides ( )--2,3-dideoxy-3-thiacytidine ( 3tc)an approved anti - human immunodeficiency virus ( hiv ) agent  and troxacitabine ( tro)an experimental anti - neoplastic agent . 
 the first step in activating these agents is catalyzed by dck . 
 our studies reveal how dck , which normally catalyzes phosphorylation of the natural d - nucleosides , can efficiently phosphorylate substrates with non - physiologic chirality . 
 the capability of dck to phosphorylate both d- and l - nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves . 
 first , the nucleoside - binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein - ligand interactions responsible for productive substrate positioning . 
 second , the pseudo - symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the l- and d - enantiomeric forms to adopt similar shapes when bound to the enzyme . 
 this is the first analysis of the structural basis for activation of l - nucleoside analogs , providing further impetus for discovery and clinical development of new agents in this molecular class . 