The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity

Abstract

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R 1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50 %. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase

“…Amino-terminal truncations dN305R1 and dN246R1 were created by trypsin and chymotrypsin cleavage of R1 (Conner et al, 1992b).…”

“…However, a striking feature of R1 from HSV-1 and HSV-2 is the presence of an additional amino-terminal region of 311 and 318 amino acids, respectively (Nikas et al, 1986). For HSV-1 at least, this N-terminal extension is unnecessary for ribonucleotide reduction (Lankinen et al, t989;Conner et al, 1992b).…”

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase

“…1 a, lane 3) were probably the result of cleavage within the junction region between the distinct functional domains of the protein. The fragments derived from the unique N terminus stain poorly with Coomassie brilliant blue (Conner et al, 1992b) and are visualized in Fig. 1 (a) by Western blotting.…”

“…N-terminal sequencing was performed on an Applied Biosystems 477A protein sequencer (Conner et al, 1992b). Ribonucleotide reductase activity assays were performed as described by Darling eta[.…”

“…Ribonucleotide reductase activity assays were performed as described by Darling eta[. (1987) with the inclusion of 0' 1 units/100 lal of ~z-macroglobulin to inhibit proteases (Conner et al, 1992b).…”