Nucleoside Binding Site of Herpes Simplex Type 1 Thymidine Kinase Analyzed by X-ray Crystallography

Proteins. 2000 Dec 1;41(4):545-53. doi: 10.1002/1097-0134(20001201)41:4<545::aid-prot110>3.0.co;2-8.

Abstract

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.

MeSH terms

  • Adenine / analogs & derivatives*
  • Adenine / chemistry
  • Adenine / metabolism
  • Adenosine / analogs & derivatives
  • Adenosine / chemistry
  • Adenosine / metabolism
  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / chemistry
  • Adenosine Monophosphate / metabolism
  • Amino Acid Substitution
  • Antiviral Agents / chemistry
  • Antiviral Agents / metabolism
  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism
  • Herpesvirus 1, Human / chemistry*
  • Herpesvirus 1, Human / isolation & purification
  • Herpesvirus 1, Human / metabolism
  • Mutation
  • Nucleosides / chemistry
  • Nucleosides / metabolism*
  • Organophosphonates*
  • Prodrugs / chemistry
  • Prodrugs / metabolism
  • Protein Structure, Tertiary
  • Stereoisomerism
  • Substrate Specificity
  • Thymidine / chemistry
  • Thymidine / metabolism
  • Thymidine Kinase / antagonists & inhibitors
  • Thymidine Kinase / chemistry*
  • Thymidine Kinase / metabolism
  • Viral Proteins / chemistry
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism
  • Water / metabolism

Substances

  • 9-(2-hydroxypropyl)adenine
  • Antiviral Agents
  • Enzyme Inhibitors
  • Nucleosides
  • Organophosphonates
  • Prodrugs
  • Viral Proteins
  • Water
  • Adenosine Monophosphate
  • adefovir
  • Thymidine Kinase
  • Adenine
  • Adenosine
  • Thymidine