Biochemical and biophysical characterization of a novel plant protein disulfide isomerase.
We recently isolated a protein disulfide isomerase (PDI) from the Rubiaceae (coffee family) plant Oldenlandia affinis (OaPDI) and demonstrated that it facilitates the production of disulfide-knotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including size-exclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers and/or higher-order oligomers were observed at increased conditions, raising the possibility that dimerization and/or oligomerization could be a mechanism to adapt to the various-sized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI.
Gruber
Christian W
CW
Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, QLD 4072, Australia.
Cemazar
Masa
M
Mechler
Adam
A
Martin
Lisandra L
LL
Craik
David J
DJ
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D002850:Chromatography, Gel; D009682:Magnetic Resonance Spectroscopy; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008970:Molecular Weight; D032067:Oldenlandia / Q000201:enzymology*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D019704:Protein Disulfide-Isomerases