Intramolecular autoinhibition of human PEX13 modulates peroxisomal import

Abstract

Targeting and import of peroxisomal proteins depends on PEX5, PEX14 and PEX13. We present a biochemical and structural characterization of the PEX13 C-terminal region. By combining NMR spectroscopy, X-ray crystallography and biochemical methods, we show that the PEX13 SH3 domain mediates intramolecular interactions with a newly identified proximal FxxxF motif and also binds to WxxxF peptide motifs from the PEX5 NTD, demonstrating evolutionary conservation of this interaction from yeast to human. Strikingly, the C-terminal FxxxF motif autoinhibits the WxxxF/Y binding surface on the PEX13 SH3 domain. This is supported by high-resolution crystal structures, which show FxxxF or WxxxF/Y binding to the same, non-canonical surface on the SH3 domain. The FxxxF motif also binds the PEX14 NTD with micromolar affinity. Surprisingly, the canonical binding surface for PxxP motifs on the human PEX13 SH3 fold does not recognize PxxP motifs in PEX14, distinct from the yeast ortholog. The dynamic network of PEX13, PEX14 and PEX5 interactions mediated by diaromatic peptide motifs fine-tunes and modulates peroxisomal matrix import in cells.

Molecular characterization of protein translocation pores

“…In this model, first, the WxxxF/Y motif of PEX5L binds to the NTD of PEX14 with strong affinity. This is then replaced by binding of the PEX13 FxxxF motif to the NTD PEX14 and PEX13 SH3 domain to the PEX5 NTD, which possibly requires large amounts of PEX13 due to their weak affinities for both PEX5 and PEX14 (Gaussmann et al 2022). Supporting this, the detection of a large molecular mass complex that exclusively consists of PEX13 or PEX13/PEX14 has been reported already (Ravindran et al 2023;Gao et al 2022;Itoh and Fujiki 2006).…”

“…This implies that it can also act as a bait component for the PEX5 receptor in the cytosol and bring it inside the matrix. Supporting this hypothesis, it was recently shown that the C-terminal SH3 domain of human PEX13 binds WxxxF/Y motifs in the PEX5L NTD (Gaussmann et al 2022). The same group also found a novel FxxxF peptide motif in the C-terminal region SH3 domain of human PEX13, whose binding has an autoinhibitory effect (Gaussmann et al 2022).…”

“…The SH3 domain of yeast Pex13 was shown to bind to the PxxP motif of Pex14. In humans, the FxxxF motif of PEX13 binds to the PEX14 NTD (Figure 1.13) (Bottger et al 2000;Douangamath et al 2002;Emmanouilidis et al 2016;Gaussmann et al 2022). Interaction studies between the PEX14 NTD and the PEX13 FxxxF motif showed that PEX13 FxxxF (KD= 2.8 µM) exhibited two-fold more binding affinity than autoinhibited state of PEX13 SH3-CTR (KD= 5.4 µM) with PEX14 NTD.…”

“…PTS1 import is reduced with deletion of the internal FxxxF motif (Gaussmann et al 2022). The binding affinities of the PEX proteins with each other also describe a model to delineate the order of their bindings.…”

“…PEX13 also binds via its SH3 domain to the NTD of PEX14. Additionally, in PEX13 a new FxxxF motifs was identified which binds to the SH3 domain of PEX13 and has an autoinhibitory effect(Gaussmann et al 2022). Created using Biorender.com…”