Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation
Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation
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DOI：
10.1073/pnas.1112838108
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发表时间：
2011-12-20
影响因子：
11.1
通讯作者：
Cutler, Sean R.
中科院分区：
文献类型：
--
作者：
Mosquna, Assaf;Peterson, Francis C.;Cutler, Sean R.
吡拉菌素抗性（PYR）1及其相关物属于可溶性脱落酸（阿坝）受体家族，当处于激动剂稳定构象时，其抑制2C型蛋白磷酸酶（PP 2C）。考虑到它们的开关样性质，我们设想稳定其激动剂结合构象的突变可用于激活体内信号传导。为了鉴定这样的突变，我们在参与阿坝或PP 2C接触的39个高度保守的残基处对PYR 1进行位点饱和诱变。对这些位点上所有741个可能的单个氨基酸取代进行了测试，以鉴定增加基础PYR 1-PP 2C相互作用的变体，这揭示了优先聚集在PYR 1的门环和C-末端螺旋中的10个残基中的激活突变。突变导致可测量的，但不完全的受体激活在体外，然而，特定的三重和四重突变体组合，促进激动剂结合的构象，如通过异源单量子相干NMR测量，并导致完全受体激活。此外，这些突变在引入不同的家族成员时保留功能性，因此可以用于解剖体内个体受体功能，这由于冗余和家族大小而存在问题。拟南芥种子中激活的PYL 2的表达通过多种措施激活阿坝信号传导：ABA调节的基因表达的调制，超休眠的诱导，以及aba 2 -1突变体中阿坝缺乏表型的抑制。我们的研究结果为PYR 1和相关阿坝受体的系统性功能获得研究奠定了基础，并表明，尽管有大量的受体，但单个受体的激活足以激活植物中的信号传导。
Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta.