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

Abstract

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 ExWΦxE(F/Y)Φ for high affinity binding to the PEX14 NTD and discuss conservation of the (di)aromatic peptide recognition by PEX14 in other species.

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif

“…7 ; Supplementary Table 4 ). These values are in agreement with previous studies in different buffer conditions 34 . Of note, amongst the eight motifs in PEX5, W4 shows the highest relative binding affinity for PEX13 SH3 and the weakest interaction with the PEX14 NTD (Fig.…”

Intramolecular autoinhibition of human PEX13 modulates peroxisomal import

“…7, Table 3 ). These values are in agreement with previous studies in different buffer conditions (Gopalswamy et al , 2022). Of note, amongst the eight (di)aromatic peptide motifs in PEX5, W4 shows the highest relative binding affinity for PEX13 SH3 and the weakest interaction with the PEX14 NTD ( Fig.…”

Molecular characterization of protein translocation pores

“…The yeast and human PEX5 mostly differ in their unstructured N-terminus (Figure 1.12). In contrast to the yeast Pex5 protein, which possesses a single binding site for the NTD of Pex14, the human PEX5 protein is characterized by the presence of a minimum of eight binding sites for PEX14 (Neuhaus et al 2014;Otera et al 2000;Saidowsky et al 2001;Gopalswamy et al 2023). Therefore, it is possible that the size and stoichiometry of complexes formed by human PEX5…”