Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein

Yelena Sterlin, Oded Pri-Tal, Gil Zimran, Sang Youl Park, Julius Ben-Ari, Jiorgos Kourelis, Inge Verstraeten, Maayan Gal, Sean R. Cutler, Assaf Mosquna*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 μm, which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.

Original languageEnglish
Pages (from-to)928-941
Number of pages14
JournalPlant Journal
Volume98
Issue number5
DOIs
StatePublished - Jun 2019
Externally publishedYes

Keywords

  • ABA signaling
  • Bet v 1
  • PYR/PYL/RCAR
  • START domain
  • abscisic acid
  • in planta
  • polyketide cyclase-like proteins
  • protein–metabolite interactions
  • technical advance
  • technical advance

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