Density functional studies on herpes simplex virus type 1 thymidine kinase–substrate interactions: The role of Tyr-172 and Met-128 in thymine fixation

Alber, Frank; Kuonen, Oliver; Scapozza, Léonardo; Folkers, Gerd; Carloni, Paolo

doi:10.1002/(sici)1097-0134(19980601)31:4<453::aid-prot11>3.0.co;2-e

Cited by 29 publications

(10 citation statements)

References 42 publications

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“…3b). This is in marked contrast to HSV1-tk where the base is sandwiched by an aliphatic and aromatic residue, Met-128 and Tyr-172, respectively, with Ile-100 also making some contacts (19,26,36). These differences in interactions with the pyrimidine ring result in the base binding in a significantly different position to that seen in the HSV1-tk structure.…”

Section: Resultsmentioning

confidence: 73%

Crystal Structure of Varicella Zoster Virus Thymidine Kinase

Bird

Ren

Wright

et al. 2003

Journal of Biological Chemistry

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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 ...

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Section: Resultsmentioning

confidence: 73%

Crystal Structure of Varicella Zoster Virus Thymidine Kinase

Bird

Ren

Wright

et al. 2003

Journal of Biological Chemistry

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“…40,41 In this respect, it has been shown that methionines display mainly hydrophobic character, 40 at least in the context of artificial hairpin peptides where their measurements were made. In fact, it could be that the water molecules are actually stabilizing the cf2 conformations rather than the cf1 conformation by binding to sulfur.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis of the Inhibition of Golgi α-Mannosidase II by Mannostatin A and the Role of the Thiomethyl Moiety in Ligand−Protein Interactions

et al. 2006

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The X-ray crystal structures of mannose trimming enzyme Drosophila Golgi α-mannosidase II (dGMII) complexed with the inhibitors mannostatin A (1) and an N-benzyl analog (2) have been determined. Molecular dynamics simulations and NMR studies have shown that the five-membered ring of mannostatin A is rather flexible occupying pseudo-rotational itineraries between 2 T 3 and 5 E, and 2 T 3 and 4 E. In the bound state, mannostatin A adopts a 2 T 1 twist envelope conformation, which is not significantly populated in solution. Possible conformations of the mannosyl oxacarbenium ion and an enzyme-linked intermediate have been compared to the conformation of mannostatin A in the co-crystal structure with dGMII. It has been found that mannostatin A best mimics the covalent linked mannosyl intermediate, which adopts a 1 S 5 skew boat conformation. The thiomethyl group, which is critical for high affinity, superimposes with the C-6 hydroxyl of the covalent linked intermediate. This functionality is able to make a number of additional polar and non-polar interactions increasing the affinity for dGMII. Furthermore, the X-ray structures show that the environment surrounding the thiomethyl group of 1 is remarkably similar to the arrangements around the methionine residues in the protein. Collectively, our studies contradict the long held view that potent inhibitors of glycosidases mimic an oxacarbenium ion like transition state.

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“…This finding was consistent with the study [32] that activity was retained only with phenylalanine at Tyr 172 of HSV-1 TK. On the basis of the information obtained from the density functional studies on HSV-1 TK, the substrate-Tyr 172 interaction is essentially an electrostatic force and was suggested to be involved in the substrate binding [48]. The three-dimensional structure of the HSV-1 TK showed the aromatic ring of the tyrosine residue at this position stacked on to the thymine ring, and Arg 163 (HSV-1 TK) formed the hydrogen bond with the hydroxy group of the tyrosine residue at this position [36].…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

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Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

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Density functional studies on herpes simplex virus type 1 thymidine kinase–substrate interactions: The role of Tyr-172 and Met-128 in thymine fixation

Cited by 29 publications

References 42 publications

Crystal Structure of Varicella Zoster Virus Thymidine Kinase

Crystal Structure of Varicella Zoster Virus Thymidine Kinase

Structural Basis of the Inhibition of Golgi α-Mannosidase II by Mannostatin A and the Role of the Thiomethyl Moiety in Ligand−Protein Interactions

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

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