The Leishmania donovani peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on peroxin 5

• Published in: Biochimica et biophysica acta Vol. 1850; no. 11; pp. 2203 - 2212
• Main Authors: Hojjat, Hamed, Jardim, Armando
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2015
• Subjects: Amino Acid Motifs; Binding Sites; biochemical pathways; biogenesis; drugs; enzyme-linked immunosorbent assay; fluorescence; fluorescence emission spectroscopy; gel chromatography; glycolysis; Glycosome; hydrophobicity; Leishmania; Leishmania donovani; mutagenesis; oxidation; Peptide Fragments - chemistry; Peroxin 14; Peroxin 5; Peroxisome-Targeting Signal 1 Receptor; Protein Stability; Protein Structure, Quaternary; protein transport; proteins; Protein–protein interaction; Protozoan Proteins - chemistry; Receptors, Cytoplasmic and Nuclear - chemistry; synthetic peptides; two hybrid system techniques; HRP; TMB; PTS; Protein–protein interaction; SEC; Peroxin 14; LdPEX; PBST; Leishmania; Peroxin 5; BACTH; ITC; Glycosome
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.07.006

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. 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. The LdPEX14 binding site was mapped to residues 280–300 on LdPEX5, a region containing the pentapeptide motif W293AQEY297. 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. 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. •LdPEX14 binds the pentapeptide motif with the consensus sequence [W/F/L]XXX[W/F/L].•The LdPEX5–LdPEX14 interaction mediated by W293AQEY297 pentapeptide motif.•LdPEX5 and LdPEX14 association is stabilized by dimer–dimer contacts.