People are susceptible to a variety of strains of the herpes simplex virus (HSV). HSV-1 and HSV-2 are particularly prevalent, and can cause cold sores (oral herpes) and genital lesions (genital herpes). Medications for herpes typically target the viral thymidine kinases (TK) which utilize ATP to phosphorylate deoxythymidine (dT) in the formation pathway of deoxythymidine triphosphate for DNA synthesis (Thymine --> Thymidine formation pathway). This treatment is appropriate as the viral TKs differ substantially from host TKs.
Ganciclovir is a potent HSV TK targeting drug that binds specifically to the viral thymidine kinase in place of dT. (Champness et al, 1998) The phosphorylated drug then competes with GTP for incorporation into replicating viral DNA, thereby stalling and terminating DNA synthesis (US Dept. of Health, 2004). In addition to its use in treating HSV, this interaction is of great interest in genetic research as it is utilized in the transformation of embryonic stem cells to make transgenic test subjects (Kimball, 2005).
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The two subunits of the thymidine kinase dimer each consist of 376 amino acids (Champness et al, 1998) and contain 15 α-helices and 7 β-sheets (Brown et al, 1995). The monomers are joined primarily by hydrophobic interactions between helices H3, H4, H7, and H13 that form a flat dimerization surface . These hydrophobic van der Waals forces act across the plane dividing the monomers and thereby stabilize the enzyme as a dimer. As can be seen, there are relatively few water molecules in the interface between the two subunits . (Champness et al, 1998).
As the subunits are identical and it has been found that the monomers are individually active, further exploration of the active binding site of thymidine kinase will be conducted using only a single subunit for ease of visualization.
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As the purpose of this protein is to phosphorylate an unphosphorylated thymidine (dT), one of the most important abilities of TK is its ability to bind ATP as a substrate and eventally catalyze the transfer of a phosphate group from ATP to thymidine. The enzyme binds to ATP via a strand-turn-helix motif at residues 48-69 near the exterior surface of the molecule. (Brown et al, 1995)
This structure serves as an anion hole in which the three phosphates of ATP can bind in complex with Mg2+ (not visualized) that is coordinated by Asp 162 at the C-terminal end of beta sheet B3 (Brown et al, 1995).
The thymidine binding site is in the center of each monomer, where helices H2, H3 and H4 form a pocket for dT .
The amino acid sequence at positions 161-192 is highly conserved among herpes simplex viruses. A central binding interaction occurs within this segment as Tyr 172 stacks with the T base. Mutations at the 172 Tyr are functional only when they lead to replacement by Phe (Brown et al, 1995).
Arg 163 binds the 5’O of dT and positions it for phosphorylation.The remaining segment (183-192) forms the H7 helix which in addition to contributing to dimerization, coordinates helices H5 and H6 via hydrophobic interactions.
The stacking interactions and van der Waals forces within the pocket orient the pyrimidine ring of dT in the plane of Gln 125 allowing for the formation of two hydrogen bonds with the 3N and 4-O – similar to the thymidine’s classic DNA base pairing. The sugar is placed in close contact with the A-helices, especially Tyr 101 , the –OH terminus of which forms a hydrogen bond with the 3’O of dT’s deoxyribose.
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Finally, thymidine kinase acts catalytically to phosphorylate thymidine , readiying it for further phosphorylation and eventual incorportation into DNA.
(Champness et al, 1998, Brown et al, 1995).
Ganciclovir binds within the dT binding pocket, with the 4’ O forming a bond with Arg 163 that resembles the bond made by the 5’ O of dT, and another OH bond occurring with Tyr 101 . Atoms N1 and O6 form hydrogen bonds with Gln 125 similar to those formed between Gln 125 and thymidine (rotated 180º). Further stabilization is provided by the interactions with His 58 , and by van der Waals interactions between Tyr 172, Met 231, Met 128, and Ile100 .
(Champness et al, 1998, Brown et al, 1995).
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Ganciclovir is one of a number of drugs used to treat HSV. Research into the binding capabilities of TK and other ligands can serve to elucidate the enzyme’s natural function, as well as provide for the development of more effective HSV treatment (Champness et al, 1998).
The TK-Ganciclovir interaction is at present used extensively in genetic research. In the process of transforming embryonic stem cells, the gene encoding TK is included in the transforming vector outside the segment of the vector that is to be inserted. Correct insertions do not incorporate the gene for TK, and the properly transformed cells are not affected by ganciclovir. Random insertions of the vector, however, retain the gene for TK, thus making the cells susceptible to the drug, and enabling cell lines with incorrectly inserted vectors to be easily eliminated. (Kimball, 2005).
Brown, D.G., Visse, R., Sandhu, G., et al. Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nat. Struct. Biol. 2:876-881, 1995.
Champness, J.N., Bennett, M.S., et al. Exploring the Active Site of Herpes Simplex Virus Type-1 Thymidine Kinase by X-Ray Crystallography of Complexes With Aciclovir and Other Ligands. PROTEINS: Structure, Function, and Genetics 32:350-361, 1998.
Kimball, J., "Transgenic Animals", users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicAnimals.html#step_1, Created May 19,2005., Accessed December 11, 2005.
US Department of Health and Human Services, "Ganciclovir",
Updated September 28, 2004. Accessed December 11, 2005.