Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display.
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.
Department of Biochemistry and Biophysics.
Gurkan
G
Guntas
Department of Biochemistry and Biophysics.
Steven M
SM
Lewis
Department of Cell Biology and Physiology Lineberger Comprehensive Cancer Center, University of North Carolina, 120 Mason Farm Road, Genetic Medicine Building 3010, Chapel Hill, NC 27599-7260, USA.
Kathleen M
KM
Mulvaney
Department of Cell Biology and Physiology Lineberger Comprehensive Cancer Center, University of North Carolina, 120 Mason Farm Road, Genetic Medicine Building 3010, Chapel Hill, NC 27599-7260, USA.
Erica W
EW
Cloer
Department of Biochemistry and Biophysics.
Ashutosh
A
Tripathy
Department of Biochemistry and Biophysics.
Thomas R
TR
Lane
Department of Cell Biology and Physiology Lineberger Comprehensive Cancer Center, University of North Carolina, 120 Mason Farm Road, Genetic Medicine Building 3010, Chapel Hill, NC 27599-7260, USA.
Michael B
MB
Major
Department of Cell Biology and Physiology Lineberger Comprehensive Cancer Center, University of North Carolina, 120 Mason Farm Road, Genetic Medicine Building 3010, Chapel Hill, NC 27599-7260, USA bkuhlman@email.unc.edu.
Brian
B
Kuhlman
D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061505:Cell Surface Display Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors*; D000072019:Kelch-Like ECH-Associated Protein 1; D008958:Models, Molecular; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011994:Recombinant Proteins
KEAP1; NRF2; Rosetta molecular modeling program; protein design; redox regulation