title: Analysis of the Leishmania peroxin 7 interactions with peroxin 5, peroxin 14 and PTS2 ligands.
abstract: 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.
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Ana Victoria C
authors.initials: AV
authors.lastname: Pilar
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Rona
authors.initials: R
authors.lastname: Strasser
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: James
authors.initials: J
authors.lastname: McLean
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Quinn
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Normand
authors.initials: N
authors.lastname: Cyr
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Hamed
authors.initials: H
authors.lastname: Hojjat
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Anwer Hasil
authors.initials: AH
authors.lastname: Kottarampatel
authors.affiliation: *Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada, H9X 3V9.
authors.forename: Armando
authors.initials: A
authors.lastname: Jardim
mesh_terms: D002135:Calcium-Binding Proteins / Q000187:drug effects*; D057927:Hydrophobic and Hydrophilic Interactions; D007891:Leishmania / Q000378:metabolism; D008024:Ligands; D008830:Microbodies / Q000378:metabolism*; D000074437:Peroxisomal Targeting Signal 2 Receptor; D015800:Protozoan Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002135:Calcium-Binding Proteins; D008024:Ligands; C470663:PEX14 protein, Leishmania donovani; D000074437:Peroxisomal Targeting Signal 2 Receptor; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C060529:cp35 protein, Columba livia