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

Abstract

Summary 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.

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

“…One unique method that overcomes these limitations and allows for metabolome-wide analysis of protein-metabolite interactions in close to in vivo conditions is affinity purification using protein as bait. Affinity purification is a commonly used technique to study proteinprotein interactions, but was recently adapted to study the interactions between proteins and small molecules, and many of the captured interactions have regulatory potential (Edwards & Dixon, 2018;Li et al, 2010;Luzarowski et al, 2017;Luzarowski, Wojciechowska, & Skirycz, 2018;Maeda et al, 2013;Sterlin et al, 2019).…”

Characterization of acyl-lipid thioesterase (ALT) enzymes from plants

“…For example, Arabidopsis gene co-expression networks indicate that the expression pattern of AtALT2 strongly overlaps with several uncharacterized genes belonging to the GDSL-like lipase/esterase, α/β hydrolase, HXXXD-type acyltransferase, lipid-transfer protein, and Sadenosyl-L-methionine-dependent methyltransferase families, which could potentially be involved in the transport or chemical modification of the β-keto FA products of AtALT2 (Obayashi et al, 2022). Interestingly, AtALT2 expression also overlaps with MLP-LIKE PROTEIN 165 (MLP165), a gene encoding a member of the multifunctional, labile major latex protein family that is known to bind acyl lipids (Sterlin et al¸ 2019). MLP165 could therefore be involved in the transport or metabolism of products that arise from AtALT2 activity (Sterlin et al, 2019).…”

“…Interestingly, AtALT2 expression also overlaps with MLP-LIKE PROTEIN 165 (MLP165), a gene encoding a member of the multifunctional, labile major latex protein family that is known to bind acyl lipids (Sterlin et al¸ 2019). MLP165 could therefore be involved in the transport or metabolism of products that arise from AtALT2 activity (Sterlin et al, 2019). Performing small-molecule pull-down assays with protein extracts from transgenic plant tissues overexpressing both AtALT2 and MLP165, similar to the experiments described by Sterlin et al, to identify ligands interacting with MLP-like proteins, could determine whether this is the case.…”