Fig. 6: D-macrocyclic peptides identified from MIPD are high-affinity ligands to CHIP.

A The E3 ubiquitin ligase CHIP marks chaperone bound client proteins for degradation through recognition of a C-terminal peptide motif (PDB ID: 4KBQ). B The TPR domain of CHIP recognizes a C-terminal IEEVD motif. C Mirror-image phage display of the TPR domain of CHIP affords three D-macrocyclic peptide binders to a common groove. X-ray co-crystal structures of the identified sequences are shown projected onto the CHIP surface from d-H202, with annotated helical directions. d-H201 is shown in yellow, d-H202 in blue, and d-H203 in purple. Lower-case letters in the sequences denote single-letter abbreviations for d-amino acids. Binding affinities were determined from competition FP of each peptide to FAM-Hsp70-peptide bound recombinant CHIP. D All three binders utilize different chemical contacts to engage the same pockets on CHIP, despite different helical directions. Interacting side-chains from the X-ray co-crystal structures of each binder are shown overlaid. Side-chain colorings are as in (C). E The discovered binders utilize unique side-chains to engage contact sites of the native peptide substrate. The X-ray co-crystal structure of d-H201 is shown overlaid with the Hsp70 recognition peptide (PDB ID: 4KBQ). Overlapping interactions are highlighted, with d-H201 in yellow and Hsp70 peptide in pink. The lower-case d inserted before a single letter amino acid abbreviation denotes d-amino acids.