RRC ID 48720
Author Rahier R, Noiriel A, Abousalham A.
Title Functional Characterization of the N-Terminal C2 Domain from Arabidopsis thaliana Phospholipase Dα and Dβ.
Journal Biomed Res Int
Abstract Most of plant phospholipases D (PLD) exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca2+-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDα from Arabidopsis thaliana (AtPLDα) in the yeast Pichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDα was found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, in E. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDα along with its equivalent C2 domain of the AtPLDβ, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca2+-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca2+. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca2+-binding site within the C2 domain of AtPLDα.
Volume 2016
Pages 2721719
Published 2016-1-1
DOI 10.1155/2016/2721719
PMID 28101506
PMC PMC5215601
MeSH Arabidopsis / enzymology* Arabidopsis Proteins / chemistry* Arabidopsis Proteins / metabolism Calcium / chemistry* Calcium / metabolism Membrane Lipids / chemistry* Membrane Lipids / metabolism Models, Molecular* Phospholipase D / chemistry* Phospholipase D / metabolism Protein Binding Protein Domains
IF 2.276
Times Cited 5
Arabidopsis / Cultured plant cells, genes pdx82318