Inhibiting the dimeric restriction endonuclease EcoRI using interfacial helical peptides.
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.
Brickner
M
M
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Chmielewski
J
J
D002850:Chromatography, Gel; D002942:Circular Dichroism; D015246:Deoxyribonuclease EcoRI / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D010455:Peptides; D015246:Deoxyribonuclease EcoRI