title: The Leishmania donovani peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on peroxin 5.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste. Anne de Bellevue, Quebec, Canada.
authors.forename: Hamed
authors.initials: H
authors.lastname: Hojjat
authors.affiliation: Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste. Anne de Bellevue, Quebec, Canada. Electronic address: armando.jardim@mcgill.ca.
authors.forename: Armando
authors.initials: A
authors.lastname: Jardim
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D010446:Peptide Fragments / Q000737:chemistry*; D000074435:Peroxisome-Targeting Signal 1 Receptor; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D015800:Protozoan Proteins / Q000737:chemistry*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*
chemical_list: C470663:PEX14 protein, Leishmania donovani; D010446:Peptide Fragments; D000074435:Peroxisome-Targeting Signal 1 Receptor; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear
keywords: Glycosome; Leishmania; Peroxin 14; Peroxin 5; Protein–protein interaction