Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit

Abstract

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.

Demonstration of segmental mobility in the functionally essential car☐yl terminal part of ribonucleotide reductase protein R2 from Escherichia coli

“…This part of R2 has an important role in the association with the R1 protein [2][3][4][5][6][7][8][9]. The NMR observable segment also contains the conserved residues, Tyr-356 and Glu-350, that are invariable between species [22].…”

“…It has been shown that the carboxyl terminal part of protein R2 is important for the binding of protein R2 to protein R1 both in the E. eoli enzyme [24] and in the mammalian and Herpes simplex type I enzymes [5][6][7][8][9]. Truncations of C-terminal residues of protein R2 impair binding of protein R1 [2], and point mutations of conserved residues in the C-terminus reduces or abolishes catalytic activity [34].…”

Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

“…Furthermore, it does not seem to be essential for catalytic activity (39). The C-terminal seven residues, which were shown to be essential for binding between the R1 and R2 proteins in the mouse RNR (35,36), show high similarity between the trypanosome and mouse R2 proteins. The only difference is that Thr-385 (mouse R2 numbering) is replaced by a serine in the T. brucei R2 protein.…”

“…This observation led to the develop-ment of potent peptidomimetic inhibitors of HSV RNR with antiviral activity in vivo (34). The inhibitory effect of R2 C-terminal peptides has also been demonstrated for mammalian (35,36) and E. coli class Ia (37) RNRs.…”

Crystallization and crystallographic investigations of the small subunit of mouse ribonucleotide reductase

“…In mouse R2, the C-terminal 7 residues are essential for subunit interactions while the C-terminal peptide with the same function in E. coli R2 is much longer [18]. The mouse C-terminal heptapeptide was shown to be highly flexible and unstructured by NMR studies, but on addition of R1 it becomes rigid and structured [19].…”