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. 2010 Aug 12;53(15):5792-800.
doi: 10.1021/jm1005379.

The sugar ring of the nucleoside is required for productive substrate positioning in the active site of human deoxycytidine kinase (dCK): implications for the development of dCK-activated acyclic guanine analogues

Saugata Hazra  1 , Manfred Konrad, Arnon Lavie
Affiliations

The sugar ring of the nucleoside is required for productive substrate positioning in the active site of human deoxycytidine kinase (dCK): implications for the development of dCK-activated acyclic guanine analogues

Saugata Hazra et al. J Med Chem. .

Abstract

The low toxicity of acyclovir (ACV) is mainly due to the fact that human nucleoside kinases have undetectable phosphorylation rates with this acyclic guanine analogue. In contrast, herpes virus thymidine kinase (HSV1-TK) readily activates ACV. We wanted to understand why human deoxycytidine kinase (dCK), which is related to HSV1-TK and phosphorylates deoxyguanosine, does not accept acyclic guanine analogues as substrates. Therefore, we crystallized dCK in complex with ACV at the nucleoside phosphoryl acceptor site and UDP at the phosphoryl donor site. The structure reveals that while ACV does bind at the dCK active site, it does so adopting a nonproductive conformation. Despite binding ACV, the enzyme remains in the open, inactive state. In comparison to ACV binding to HSV1-TK, in dCK, the nucleoside base adopts a different orientation related by about a 60 degrees rotation. Our analysis suggests that dCK would phosphorylate acyclic guanine analogues if they can induce a similar rotation.

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Figures

Figure 1
Figure 1
Binding of ACV to human dCK. A. Chemical structure of dG and its acyclic analogs GCV and ACV. B. Parallel and perpendicular views of ACV as bound in monA of the dCK+ACV+UDP structure. The terminal hydroxyl group in ACV that corresponds to the ribose 5′-hydroxyl is labeled. C. Analogous views of ACV from monB. D. Analogous views of ACV from monD. E. Similar to the situation observed in the dCK+ACV complex structure, in the HSV1-TK+ACV complex structure, ACV adopts multiple conformations. Shown are the parallel and perpendicular views of ACV in conformation 1. F. Analogous views of ACV in conformation 2. G. For reference, shown are the analogous views of dG from the dCK+dG+UDP structure.
Figure 2
Figure 2
A ball-and-stick representation (A) and a schematic representation (B) of ACV binding to monB. Note the 4.4 Å distance between the side chain of Glu53 and the terminal hydroxyl group of ACV. Such a long distance precludes the activation of the nucleoside to attack the phosphoryl donor.
Figure 3
Figure 3
Structure-based sequence alignment of human nucleoside kinases of the same family (deoxycytidine kinase (hdCK); deoxyguanosine kinase (hdGK); mitochondrial thymidine kinase 2 (hTK2)), drosophila nucleoside kinase (dNK) that is able to phosphorylate all four nucleosides, and thymidine kinase from herpes virus, type I (HSV1- TK). Color scheme: orange for residues conserved in all 5 sequences; yellow when at least 3 of the 5 residues are identical; the non-identical residues in such cases are also colored yellow if homologous. The P-loop and insert regions are indicated, as are select critical residues based on the dCK numbering. E53 – acts to activate the 5′-hydroxyl group; W58 and Y86 – indicators of the open and closed enzyme states; Q97 – binds the base of the nucleoside; R104 and D133 are residues we have identified to be responsible for the lack of thymidine kinase activity by dCK; R128, a part of the ERS motif, activates E53; R188 – involved in binding the nucleotide donor; R192/R194 – in the Lid region, play a role in stabilizing the transition state; E197 – seems to play a role in stabilizing the closed state by directly interacting with the 3′-hydroxyl group of the nucleoside.
Figure 4
Figure 4
ACV binds to dCK but does not elicit the transition to the closed enzyme state. A. Overlay of dCK in complex with ACV mon B (light green) and dA (blue; PDB ID 2ZI6). The nucleosides are at the center of the image, UDP at the right. The good fit between the structures shows that ACV binds to the same open enzyme state as observed previously in the complex structure of dCK with dA+UDP. For visual clarity only a single monomer of the dimeric dCK is shown. B. Overlay of dCK in complex with ACV mon B (light green), and dG (light blue; PDB ID 2ZI7). In the dCK complex with dG+UDP, the enzyme adopts the closed state. Arrows point to regions where the differences in enzyme conformation are most apparent. C. A close-up of the overlay of ACV monB (light green) and dG (light blue). Note the different conformation of Trp58 and Tyr86, which are indicators of the enzyme conformation ‘open’ in the case of the ACV complex, ‘closed’ in the dG complex. Zoom on the nucleosides (right side) demonstrates the difference of guanine base orientation between the two complexes. To aid in seeing this difference, a gray sphere highlights the guanine amino group at the 2-position. Note how the bases are related by a relative rotation of ~180 degrees.
Figure 5
Figure 5
Upon binding to HSV1-TK, ACV achieves the critical close contact between the terminal hydroxyl group and the activating glutamic acid. Shown is an overlay of HSV1-TK in complex with thymidine (red; PDB ID 1KIM) and ACV (yellow; PDB ID 2KI5). ACV binds in two conformations, labeled conf1 (orange) and conf2 (yellow). Gray spheres indicate the positions of the hydroxyls that would become phosphorylated. Note that, both in the case of dT and ACV-conf1 binding, a close contact is achieved between the terminal hydroxyl group and the side chain of Glu83. Therefore, the carboxylic acid can activate either nucleoside to attack the γ-phosphate of the phosphoryl donor. Inset: side view parallel to the base of the nucleosides. While the position of the guanine base is similar to that of the thymine base, they are not totally parallel.
Figure 6
Figure 6
Modeling dG binding to HSV1-TK. A. We superimposed a dG molecule (light blue) on the ACV molecule based on the common guanine base, using the HSV1-TK ACV structure (yellow). ACV makes positive interactions with Glu83 and Arg178 – green arrows. Were dG to bind in the same position, the ribose ring would be too close to Tyr101 (red arrow). Hence, dG cannot bind to HSV1-TK in a similar fashion as ACV. B. Overlay of the HSV1-TK structure in complex with dT (red) on the dCK structure in complex with dG (light blue). Arg176 would act to repel dG due to the unfavorable interactions with the guanine amino group (red arrow), and would also lack the positive interaction present in dCK between Arg104 and the guanine carbonyl group (green arrow). These factors suggest the reasons that make dG a very poor HSV1-TK substrate.
Figure 7
Figure 7
Contrasting ACV binding to dCK (light green) and HSV1-TK (yellow). For clarity, only the ACV conformation that brings the terminal hydroxyl group closer to the catalytic glutamic acid is shown. In both enzymes, the guanine base is in the same location, but different by a relative rotation of ~60 degrees. The bidentate interaction between Arg176 of HSV1-TK and ACV cannot be made by dCK, where the homologous residue is Leu141. The relative rotation of the nucleoside in HSV1-TK positions the terminal hydroxyl group within 3.3 Å to Glu83. Lacking this interaction in dCK, the terminal hydroxyl is 4.4 Å from Glu53. Thus, Glu53 is too far to be able to activate the hydroxyl group for attack on the phosphoryl donor, revealing why ACV is not phosphorylated by dCK. Inset: The plane of the guanine base is slightly off-set between its position in dCK and in HSV1-TK.
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