脱落酸（Abscisic acid，ABA）是植物体内最重要的激素之一，它可以调控植物生长发育，同时提高植物抗旱、耐盐等对逆境的反应能力。关于 ABA 受体的研究是 ABA 信号通路研究领域的核心问题。 2009 年，两个科研小组同时报道了拟南芥 PYR/PYL/RCAR 蛋白家族为 ABA 的潜在受体。我组解析了 apo-PYL2、 ABA-PYL2、 ABA-PYL1-ABI1 三个高分辨率的晶体结构。结合生物化学实验数据，证明了 PYL 蛋白家族为拟南芥的 ABA 受体，且揭示了 ABA 介导的 PYL 对 PP2C 去磷酸化酶活性的抑制机理。结构显示，PYL2 以同源二聚体存在，在结合 ABA 之后，PYL2 中柔性区域 CL2 loop 变为闭合状态，同时二聚化界面被削弱，这种构象改变介导了对 PP2C 的识别和抑制过程。作为ABA 的选择性激动剂，pyrabactin 与 ABA 化学结构相差甚远，但它也可通过 PYL抑制 PP2C 活性。我们通过解析 pyrabactin-PYL1 的复合物结构，发现了 ABA 与pyrabactin 在结合 PYL1 上的共性，提出了“电磁铁”模型：小分子发挥效应需具有大的疏水模块，通过疏水作用力使 CL2 闭合；同时需具有极性模块，与空腔内极性氨基酸相互作用进而被锚定。不同于 ABA，pyrabactin 对 PYL 具有选择性，通过进一步解析 pyrabactin-PYL2 的结构，我们发现 pyrabactin 在 PYL1与 PYL2中以不同的方位存在，结合生化试验，证明仅因一个氨基酸的不同（Valine 与Isoleucine）便导致 PYL1 与 PYL2 对 pyrabactin 需使用不同的结合方式，从而决定了 pyrabactin 的选择性。 为进一步探讨 PYL 介导的 ABA 信号通路的复杂机制， 我们系统检验了拟南芥中 10 种 PYL 在体外对 4 种 PP2C 活性的影响。结果表明， 一类 PYL 亚家族（以 PYL10 为代表）可独立于 ABA 抑制 PP2C 活性，且PYL10 在溶液中可直接与 PP2C 形成稳定复合物。 为解释其分子机制， 我们解析了 apo-PYL10和 PYL10-HAB1 复合物结构，结合生化数据发现了 PYL 独立于 ABA 发挥作用需具备两个条件：在溶液中以单体形式存在；小分子结合腔入口处需有大的疏水氨基酸。经过对 ABA 受体 PYL 蛋白一系列结构的解析和系统的生化分析， 解决了 PYL与 ABA 的结合及对下游信号通路调节的机制。同时对 PYL 家族蛋白进行分类，为解释了拟南芥中 PYL 的冗余和复杂的调控机制提供帮助。对 pyrabactin 的研究为设计 ABA 功能类似物并应用于农业生产提供了指导。
Abscisic acid (ABA), one of the most essential phytohormones in plants, plays a key role in regulating various developmental processes and improving stress resistance to drought and salt. ABA receptors are at the core of the research of ABA signaling pathway. In 2009, two research groups reported a potential soluble ABA receptor PYR/PYL/RCAR protein family in Arabidopsis.Using X-ray crystallography, we solved the structures of apo-PYL2, ABA-PYL2 and ABA-PYL1-ABI1 at high atomic resolution. Combined with biochemical analysis, we found PYLs are ABA receptors and revealed an ABA-mediated inhibition mechanism towards PP2Cs, a family of phosphatase. ABA’s binding leads to a remarkable conformational change on CL2 loop and weakens PYL2’s dimer interface, which helps to recognize PP2Cs and inhibit its activity. The synthetic compound pyrabactin, whose chemical structure differs a lot from ABA, could function through PYLs. In order to understand the mechanism, we solved the crystal structure of pyrabactin-PYL1 and proposed an “ectromagnet” model for the ligand perception of PYLs: the ligand should contain a hydrophobic module that can close CL2 loop and a polar module to be anchored in the ligand binding pocket. Unlike ABA, pyrabactin only binds to a selective pool of PYLs. To understand what determines the selectivity, we determined the structure of pyrabactin-PYL2 and found that pyrabactin resides in distinct approaches compared with that in PYL1, which is caused by a single amino acid alternation between Valine and Isoleucine. To delineate the complex regulation by PYLs of ABA signaling pathway, we systematically assessed the inhibitory effect of ten PYLs on four PP2Cs. The results showed that one subfamily represented by PYL10 inhibits PP2Cs without ABA. Through solving apo-PYL10 and PYL10-HAB1 complex structure, we found out that the constitutively active PYLs are monomeric and contain a bulky hydrophobic residue guarding the ligand-binding pocket.Our studies on PYLs help us to classify PYLs to provide a framework to understand PYL’s complex regulation. The insights we gained form examination of pyrabactin provide us with a general principle to design specific ABA agonists that are beneficial in the agricultural field.
