Nucleoside binding site of Herpes simplex type 1 thymidine kinase analyzedby X-ray crystallography

The crystal structures of the full-length Herpes simplex virus type 1 thymi dine kinase in its unligated form and in a complex with an adenine analogue have been determined art 1.9 Angstrom resolution. The unligated enzyme con tains four water molecules in the thymidine pocket and reveals a small indu ced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thym ine and guanine analogues have been reported. The adenine analogue constitu tes a new lead compound for enzyme-prodrug gene therapy. In addition, the s tructure of mutant Q125N modifying the binding site of the natural substrat e thymidine in complex with this substrate has been established at 2.5 Angs trom 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 hydrog en blond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutan t structure represents a first step toward improving the virus-directed enz yme-prodrug gene therapy by enzyme engineering. Proteins 2000;41:545-553. ( C) 2000 Wiley-Liss, Inc.