RRC ID 63980
Author Pokotylo I, Hellal D, Bouceba T, Hernandez-Martinez M, Kravets V, Leitao L, Espinasse C, Kleiner I, Ruelland E.
Title Deciphering the Binding of Salicylic Acid to Arabidopsis thaliana Chloroplastic GAPDH-A1.
Journal Int J Mol Sci
Abstract Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.
Volume 21(13)
Published 2020-6-30
DOI 10.3390/ijms21134678
PII ijms21134678
PMID 32630078
PMC PMC7370300
MeSH Amino Acid Sequence Arabidopsis / enzymology* Binding Sites Chloroplasts / enzymology Glyceraldehyde-3-Phosphate Dehydrogenases / chemistry Glyceraldehyde-3-Phosphate Dehydrogenases / genetics Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism* Molecular Docking Simulation Molecular Dynamics Simulation NAD Point Mutation Salicylic Acid / metabolism*
IF 4.556
Arabidopsis / Cultured plant cells, genes pda03105