Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display

Gurkan Guntas, Steven M. Lewis, Kathleen M. Mulvaney, Erica W. Cloer, Ashutosh Tripathy, Thomas R. Lane, Michael B. Major, Brian Kuhlman

Research output: Contribution to journalArticle

  • 6 Citations

Abstract

In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

LanguageEnglish (US)
Pages1-9
Number of pages9
JournalProtein Engineering, Design and Selection
Volume29
Issue number1
DOIs
StatePublished - Sep 25 2015

Fingerprint

Bacteriophages
Oxidation-Reduction
Cysteine
Display devices
Proteins
Transcription factors
Ubiquitination
Transcription
Binders
Transcription Factors
Genes
Chemical activation
Cells
Degradation
Molecules

Keywords

  • KEAP1
  • NRF2
  • protein design
  • redox regulation
  • Rosetta molecular modeling program

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Cite this

Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display. / Guntas, Gurkan; Lewis, Steven M.; Mulvaney, Kathleen M.; Cloer, Erica W.; Tripathy, Ashutosh; Lane, Thomas R.; Major, Michael B.; Kuhlman, Brian.

In: Protein Engineering, Design and Selection, Vol. 29, No. 1, 25.09.2015, p. 1-9.

Research output: Contribution to journalArticle

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