Analysis of the Leishmania Peroxin 7 Interactions with Peroxin 5, Peroxin 14 and PTS2 Ligands

The Leishmania receptorperoxin PEX7 (LPEX7) is essential for targeting newly synthesized proteins with a PTS2 import signal into the glycosome. Here we describe the biophysical characterization of a functional LPEX7 isolated from E. coli inclusion bodies. Pull down assays showed that this LPEX7 binds the interacting partners LdPEX5 and LdPEX14, but more importantly, this receptor can specifically binds PTS2 cargo proteins in the monomeric and dimeric state. 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 8-anilino-1-naphthalenesulfonic 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 ...
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Biomolecules Source Type: research