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Trafficking of peroxisomal targeting signal 1 (PTS1) proteins to the Leishmania glycosome is dependent on the docking of the LdPEX5 receptor to LdPEX14 on the glycosomal membrane. A combination of deletion and random mutagenesis was used to identify residues in the LdPEX14 N-terminal region that are critical for mediating the LdPEX5–LdPEX14 interaction. These studies highlighted residues 35–75 on ldpex14 as the core domain required for binding LdPEX5. Single point mutation within this core domain generally did not affect the ldpex5-(203–391)–ldpex14-(1–120) interaction; notable exceptions were substitutions at Phe40, Val46 or Phe57 which completely abolished or increased the apparent Kd value for ldpex5-(203–391) binding 30-fold. Biochemical studies revealed that these point mutations did not alter either the secondary or quaternary structure of LdPEX14 and indicated that the latter residues were critical for stabilizing the LdPEX5–LdPEX14 interaction.

LPEX7 (Leishmania peroxin 7) is essential for targeting newly synthesized proteins with a PTS2 (peroxisome-targeting signal type 2) import signal into the glycosome. In the present paper, we describe the biophysical characterization of a functional LPEX7 isolated from Escherichia coli inclusion bodies. Pull-down assays showed that LPEX7 binds the interacting partners LdPEX5 (Leishmania donovani peroxin 5) and LdPEX14, but, more importantly, this receptor can specifically bind PTS2 cargo proteins in the monomeric and dimeric states. However, in the absence of interacting partners, LPEX7 preferentially adopts a tetrameric structure. Mapping studies localized the LdPEX5- and LdPEX14-binding sites to the N-terminal portion of LPEX7. Deletion of the first 52 residues abolished LdPEX14 association without altering the LdPEX5 interaction. Intrinsic fluorescence techniques suggested that each LPEX7 subunit has a single unique binding site for each of the respective interacting partners LdPEX5, LdPEX14 and PTS2 cargo proteins. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) demonstrated that LPEX7 contains a surface-exposed hydrophobic region(s) that was not altered by the binding of a PTS2 protein or LdPEX5. However, in the presence of these ligands, the accessibility of the hydrophobic domain was dramatically restricted, suggesting that both ligands are necessary to induce notable conformational changes in LPEX7. In contrast, binding of LdPEX14 did not alter the hydrophobic domain on LPEX7. It is possible that the hydrophobic surfaces on LPEX7 may be a crucial characteristic for the shuttling of this receptor in and out of the glycosome.

Background The glycosome is a unique organelle found in Kinetoplastids known to compartmentalize vital metabolic pathways including glycolysis, β-fatty acid oxidation and purine salvage. Organelle biogenesis depends on a network of proteins for trafficking and translocation of nascent protein into the glycosome. The interaction of the proteins LdPEX14 and LdPEX5 at the glycosome membrane is crucial for targeting proteins into this organelle. Methods Deletion mutagenesis, pull-down, and bacterial two hybrid assay were used to map the LdPEX5 domain bound by LdPEX14. ELISA assays, ITC, intrinsic fluorescence and size exclusion chromatography to monitor binding and structural changes associated with the LdPEX5–LdPEX14 interaction. Results and conclusions The LdPEX14 binding site was mapped to residues 280–300 on LdPEX5, a region containing the pentapeptide motif W 293 AQEY 297 . Deletion of this region abolished the LdPEX5–LdPEX14 interaction. Intrinsic fluorescence spectroscopy suggests that the stabilization of the LdPEX5–LdPEX14 complex is dependent on W293 docking into a hydrophobic pocket within the binding domain of ldpex14. Studies using a panel of synthetic peptides suggest a critical role for Y297 and to a lesser extent E296 in stabilizing the LdPEX5–LdPEX14 association. General significance We show that the LdPEX14 binding site is more promiscuous and in contrast to other eukaryotic systems will accommodate a more degenerate pentapeptide motif with the sequences WXXXW or FXXXF, findings which may be exploited for potential drug design.