Crystal Structure of Varicella Zoster Virus Thymidine Kinase

Abstract

Herpes virus thymidine kinases are responsible for the activation of nucleoside antiviral drugs including (E)-5-(2-bromovinyl)-2-deoxyuridine. Such viral thymidine kinases (tk), beside having a broader substrate specificity compared with host cell enzymes, also show significant variation in nucleoside phosphorylation among themselves. We have determined the crystal structure of Varicella zoster virus (VZV, human herpes virus 3) thymidine kinase complexed with (E)-5-(2-bromovinyl)-2-deoxyuridine 5-monophosphate and ADP. Differences in the conformation of a loop region (residues 55-61) and the position of two ␣-helices at the subunit interface of VZV-tk compared with the herpes simplex virus type 1 (human herpes virus 1) enzyme give rise to changes in the positioning of residues such as tyrosine 66 and glutamine 90, which hydrogen bond to the substrate in the active site. Such changes in combination with the substitution in VZV-tk of two phenylalanine residues (in place of a tyrosine and methionine), which sandwich the substrate pyrimidine ring, cause an alteration in the positioning of the base. The interaction of the (E)-5-(2-bromovinyl)-2-deoxyuridine deoxyribose ring with the protein is altered by substitution of tyrosine 21 and phenylalanine 139 (analagous to herpes simplex virus type 1 histidine 58 and tyrosine 172), which may explain some of the differences in nucleoside sugar selectivity between both enzymes. The altered active site architecture may also account for the differences in the substrate activity of ganciclovir for the two thymidine kinases. These data should be of use in the design of novel antiherpes and antitumor drugs.Varicella zoster virus (VZV 1 ; human herpes virus 3) is the causative agent for both a primary (chicken pox or Varicella) and a secondary disease (shingles or zoster), the latter results from activation of the latent virus. Whereas Varicella is generally considered to be a mild childhood disease, in adults it may be complicated by pneumonia and encephalitis and in immunocompromised patients it is associated with significant morbidity and mortality (1).The mammalian thymidine kinase is part of the pyrimidine salvage pathway and catalyzes the transfer of the ␥-phosphoryl group from ATP to thymidine to give thymidine monophosphate. Both HSV1-tk (human herpes virus 1-tk) and VZV-tk have an additional thymidylate kinase activity that converts thymidine monophosphate to thymidine diphosphate. The substrate specificity of VZV-tk is also broader than the host cell kinase with a wide range of nucleoside analogues able to be phosphorylated (2-4). Many of the drugs utilized to treat herpes virus infections are nucleoside analogues such as the pyrimidine nucleoside BVDU or the guanine nucleoside analogues, aciclovir and ganciclovir (Scheme 1), which are activated by the viral thymidine kinase (4).Thus, the broad substrate specificity of herpes virus thymidine kinases is of interest pharmaceutically, both for the action of antiviral drugs and increasingly for their potential use in enzyme ...

In Vitro -Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

“…The lid region has been shown to be flexible in HSV-1 TK to adopt open and closed conformations. However, the equivalent region in VZV TK (residues 184 to 193) is largely disordered, consistent with the presence of the product or an open complex where the release of ADP can be facilitated following catalysis (7).…”

“…The model of VZV TK bearing mutations associated with drug resistance was constructed using ArgusLab (Planaria Software LLC, Seattle, WA), based on the crystal structure of the VZV TK complexed to BVdUMP and ADP (Protein Data Bank [PDB] code 1OSN) (7).…”

Structures of thymidine kinase 1 of human and mycoplasmic origin

“…Herpes virus 1-TK was the first member of the family for which the structure was determined (14,15). Other members of the dNK family are generally smaller but have similar core structures, and their different substrate specificity can be related to a few differences at the active site (12,(16)(17)(18)(19). TK1 has sequence similarities to the dNKs in a P loop but lacks other significant conserved sequence motifs.…”

Crystal structures of Staphylococcus aureus type I dehydroquinase from enzyme turnover experiments

“…The use of enzyme turnover for crystallization has again been demonstrated as a useful technique, opening up new regions of "crystallization-space" that can give rise to crystals suitable for structure determination where previous methods failed. 16 …”