Open AccessExploring the binding of rationally engineered tandem-repeat proteins to E3 ubiquitin ligase Keap1
Author(s) -
Sarah K. Madden,
Laura S. Itzhaki
Publication year - 2021
Publication title -
protein engineering, design and selection
Language(s) - English
Resource type - Journals
eISSN - 1741-0134
pISSN - 1741-0126
DOI - 10.1093/protein/gzab027
Subject(s) - ubiquitin ligase , peptide , ubiquitin , dna ligase , chemistry , biochemistry , keap1 , microbiology and biotechnology , computational biology , biology , enzyme , transcription factor , gene
The process of displaying functional peptides by ‘grafting’ them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display.