Optimized small‐molecule pull‐downs define MLBP 1 as an acyl‐lipid‐binding protein
Optimized small‐molecule pull‐downs define MLBP 1 as an acyl‐lipid‐binding protein
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优化的小分子下拉将 MLBP 1 定义为酰基脂质结合蛋白
DOI：
10.1111/tpj.14272
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Mosquna, Assaf
中科院分区：
文献类型：
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作者：
Sterlin, Yelena;Pri‐Tal, Oded;Zimran, Gil;Park, Sang‐Youl;Ben‐Ari, Julius;Kourelis, Jiorgos;Verstraeten, Inge;Gal, Maayan;Cutler, Sean R.;Mosquna, Assaf
脱落酸（阿坝）受体属于START结构域超家族，其包括存在于所有生命王国中的配体结合蛋白。START结构域蛋白含有一个中心结合口袋，根据蛋白质的不同，它可以将配体结合与催化、转运或信号传导功能偶联。在拟南芥中，最具特征的START结构域蛋白是14 PYR/PYL/RCAR阿坝受体，而超家族的其他成员没有指定的配体。为了解决这个问题，我们使用亲和纯化的生物素化的蛋白质瞬时表达在烟草本塞曼耦合到非靶向LC-MS，以确定候选的结合配体。我们使用ABA-PYL相互作用优化了该方法，并表明阿坝与野生型PYL 5共纯化，但不与结合位点突变体共纯化。PYL 5对阿坝的Kd值为1.1 μm，表明该方法对许多配体-蛋白质相互作用具有足够的灵敏度。使用这种方法，我们调查了一组37个START结构域相关蛋白，从而鉴定出与MLBP 1（At 4G 01883）或MLP 165（At 1G 35260）共纯化的配体。代谢物鉴定和真实标准品的使用显示MLBP 1与单亚麻酸结合，我们使用重组MLBP 1证实了这一点。单亚麻酸还与从转基因拟南芥中纯化的MLBP 1共纯化，表明相互作用发生在天然环境中。因此，这种相对简单的方法的部署使我们能够定义蛋白质-代谢物相互作用，并更好地了解植物中蛋白质-配体相互作用。
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 inNicotiana benthamianacoupled 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. TheKdof 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.