title: Leishmania donovani peroxin 14 undergoes a marked conformational change following association with peroxin 5.
abstract: The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.
authors: Institute of Parasitology, Macdonald Campus of McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9.
authors: Normand
authors: N
authors: Cyr
authors: Kleber P
authors: KP
authors: Madrid
authors: Rona
authors: R
authors: Strasser
authors: Mark
authors: M
authors: Aurousseau
authors: Ron
authors: R
authors: Finn
authors: Juan
authors: J
authors: Ausio
authors: Armando
authors: A
authors: Jardim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002942:Circular Dichroism; D007893:Leishmania donovani / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D010447:Peptide Hydrolases