Modulation of the Leishmania donovani peroxin 5 quaternary structure by peroxisomal targeting signal 1 ligands.
The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.
Institute of Parasitology, McGill University, Montreal, Quebec, Canada.
Kleber P
KP
Madrid
Gregory
G
De Crescenzo
Shengwu
S
Wang
Armando
A
Jardim
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007893:Leishmania donovani / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; C470663:PEX14 protein, Leishmania donovani; D010455:Peptides; D000074435:Peroxisome-Targeting Signal 1 Receptor; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear