IDENTIFICATION OF STRUCTURAL DOMAINS WITHIN THE LARGE SUBUNIT OF HERPES-SIMPLEX VIRUS RIBONUCLEOTIDE REDUCTASE

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide re ductase is a bifunctional protein consisting of a unique N-terminal pr otein kinase domain and a ribonucleotide reductase domain. Previous st udies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K an d 53K, within the reductase domain. The two fragments, which were prod uced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotid e reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 mu g/ml) of trypsin or V8 pr otease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrat ions (1 mu g/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtaine d using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was inve stigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies wh ich we isolated and epitope mapped for that purpose. The trypsin cleav age site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reducta se region of HSV R1 exists in a two domain structure, and that the int erdomain linking region is protected by the unique N terminus.