Inhibiting the dimeric restriction endonuclease EcoRI using interfacial helical peptides

Abstract

Background: Many enzymes are active only in a dimeric form, including a variety of type II restriction endonucleases. Disruption of subunit interactions is therefore a potential method for multimeric enzyme inhibition. EcoRI is a homodimeric restriction endonuclease, the dimeric interface of which consists of a four-helix bundle. We set out to design helical peptides to interact with this interface and block dimer formation, thus rendering EcoRI inactive.

Results: Here we describe two synthetic, helical peptides based on the interfacial region of EcoRI. Both peptides inhibit the enzyme, but the peptide derived from the alpha 4 helix of EcoRI had both a higher helical content and better efficacy than a variant peptide, alpha 4(Leu), that has three Ile-->Leu mutations (IC50 values of 27 microM and 90 microM, and helical contents of 29% and 10%, respectively). Size-exclusion chromatography confirmed that the alpha 4 peptide disrupted dimerization of EcoRI, and circular dichroism indicated that EcoRI remained folded upon binding to alpha 4. Inhibition with alpha 4 and alpha 4(Leu) was shown to be specific for EcoRI, as the dimeric restriction enzyme PvuII was not affected by the peptides.

Conclusions: Interfacial peptide inhibitors of the dimeric EcoRI were obtained that both inhibit dimerization and endonuclease activity. The peptide sequence with a preference for a helical conformation was a more effective inhibitor, presumably because the more preorganized state enhanced interactions with the helical interface of EcoRI. The specific nature of this endonuclease-peptide interaction was also confirmed. The potential of this strategy for inhibiting other enzyme classes is currently being addressed.