INTEGRATED HOMOLOGY MODELING AND X-RAY STUDY OF HERPES-SIMPLEX VIRUS-I THYMIDINE KINASE - A CASE-STUDY

Knowledge-based homology modelling together with site-directed mutagen esis, epitope and conformational mapping is an approach to predict the structures of proteins and for the rational design of new drugs. In t his study we present how this procedure has been applied to model the structure of herpes simplex virus type 1 thymidine kinase (HSV1 TK, HS V1 ATP-thymidine-5'-phosphotransferase, EC 2.7.1.21). We have used, an d evaluated, several secondary structure prediction methods, such as t he classical one based on Chou and Fastman algorithm, neural networks using the Kabsch and Sander classification, and the PRISM method. We h ave validated the algorithms by applying them to the porcine adenylate kinase (ADK), whose three-dimensional structure is known and that has been used for the alignment of the TKs as well. The resulting first m odel of HSV1-TK consisted of the first beta-strand connected to the ph osphate binding loop and its subsequent alpha-helix, the fourth beta-s trand connected to the conserved FDRH sequence and two alpha-helix wit h basic amino acids. The 3D structure was built using the X-ray struct ure of ADK as template and following the general procedure for homolog y modelling. We extended the model by means of COMPOSER, an automatic process for protein modelling. Site-directed mutagenesis was used to e xperimentally verify the predicted active-site model of HSV1-TK. The d ata measured in our lab and by others support the suggestion that the FDRH motif is part of the active site and plays an important role in t he phosphorylation of substrates. The structure of HSV1 TK, recently s olved in collaboration with Prof G. Schulz at 2.7 Angstrom resolution, includes 284 of 343 residues of the N-terminal truncated TK. The seco ndary structures could be clearly assigned and fitted to the density. The comparison between crystallographically determined structure and t he model shows that nearly 70% of the HSV1 TK structure has been corre ctly modelled by the described integrated approach to knowledge based ligand protein complex structure prediction. This indicate that comput er assisted methods, combined with ''manual'' correction both for alig nment and 3D construction are useful and can be successful.