posted on 2021-03-08, 21:04authored byPaula
C. Ortet, Samantha N. Muellers, Lauren A. Viarengo-Baker, Kristina Streu, Blair R. Szymczyna, Aaron B. Beeler, Karen N. Allen, Adrian Whitty
Macrocycles, including macrocyclic
peptides, have shown promise
for targeting challenging protein–protein interactions (PPIs).
One PPI of high interest is between Kelch-like ECH-Associated Protein-1
(KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided
by X-ray crystallography, NMR, modeling, and machine learning, we
show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be
recapitulated in a cyclic 7-mer peptide, c[(D)-β-homoAla-DPETGE].
This compound was identified from the Nrf2-derived linear peptide
GDEETGE (KD = 4.3 μM) solely by
optimizing the conformation of the cyclic compound, without changing
any KEAP1 interacting residue. X-ray crystal structures were determined
for each linear and cyclic peptide variant bound to KEAP1. Despite
large variations in affinity, no obvious differences in the conformation
of the peptide binding residues or in the interactions they made with
KEAP1 were observed. However, analysis of the X-ray structures by
machine learning showed that locations of strain in the bound ligand
could be identified through patterns of subangstrom distortions from
the geometry observed for unstrained linear peptides. We show that
optimizing the cyclic peptide affinity was driven partly through conformational
preorganization associated with a proline substitution at position
78 and with the geometry of the noninteracting residue Asp77 and partly
by decreasing strain in the ETGE motif itself. This approach may have
utility in dissecting the trade-off between conformational preorganization
and strain in other ligand–receptor systems. We also identify
a pair of conserved hydrophobic residues flanking the core DxETGE
motif which play a conformational role in facilitating the high-affinity
binding of Nrf2 to KEAP1.