Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14

The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) in the cytosol, the PEX5/cargo complex docks at the peroxisomal membrane by binding to PEX14. The PEX14 N-terminal domain (NTD) recognizes (di)aromatic peptides, mostly corresponding to Wxxx(F/Y)-motifs, with nano-to micromolar affinity. Human PEX5 possesses eight of these conserved motifs distributed within its 320-residue disordered N-terminal region. Here, we combine biophysical (ITC, NMR, CD), biochemical and computational methods to characterize the recognition of these (di)aromatic peptides motifs and identify key features that are recognized by PEX14. Notably, the eight motifs present in human PEX5 exhibit distinct affinities and energetic contributions for the interaction with the PEX14 NTD. Computational docking and analysis of the interactions of the (di)aromatic motifs identify the specific amino acids features that stabilize a helical conformation of the peptide ligands and mediate interactions with PEX14 NTD. We propose a refined consensus motif ExW phi xE(F/Y)phi for high affinity binding to the PEX14 NTD and discuss conservation of the (di)aromatic peptide recognition by PEX14 in other species.

Automated summary

The cycling import receptor PEX5 and its binding partner PEX14 play important roles in the peroxisomal import machinery. They recognize cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) and dock at the peroxisomal membrane. This study characterizes the recognition of (di)aromatic peptides motifs by PEX14 and identifies key features involved in the interaction. The findings provide a refined consensus motif for high affinity binding to PEX14 and suggest conservation of the (di)aromatic peptide recognition by PEX14 in other species.