Data_Sheet_1_Kinase Function of Brassinosteroid Receptor Specified by Two Allosterically Regulated Subdomains.pdf

Plants acquire the ability to adapt to the environment using transmembrane receptor-like kinases (RLKs) to sense the challenges from their surroundings and respond appropriately. RLKs perceive a variety of ligands through their variable extracellular domains (ECDs) that activate the highly conserved intracellular kinase domains (KDs) to control distinct biological functions through a well-developed downstream signaling cascade. A new study has emerged that brassinosteroid-insensitive 1 (BRI1) family and excess microsporocytes 1 (EMS1) but not GASSHO1 (GSO1) and other RLKs control distinct biological functions through the same signaling pathway, raising a question how the signaling pathway represented by BRI1 is specified. Here, we confirm that BRI1-KD is not functionally replaceable by GSO1-KD since the chimeric BRI1-GSO1 cannot rescue bri1 mutants. We then identify two subdomains S1 and S2. BRI1 with its S1 and S2 substituted by that of GSO1 cannot rescue bri1 mutants. Conversely, chimeric BRI1-GSO1 with its S1 and S2 substituted by that of BRI1 can rescue bri1 mutants, suggesting that S1 and S2 are the sufficient requirements to specify the signaling function of BRI1. Consequently, all the other subdomains in the KD of BRI1 are functionally replaceable by that of GSO1 although the in vitro kinase activities vary after replacements, suggesting their functional robustness and mutational plasticity with diverse kinase activity. Interestingly, S1 contains αC-β4 loop as an allosteric hotspot and S2 includes kinase activation loop, proposedly regulating kinase activities. Further analysis reveals that this specific function requires β4 and β5 in addition to αC-β4 loop in S1. We, therefore, suggest that BRI1 specifies its kinase function through an allosteric regulation of these two subdomains to control its distinct biological functions, providing a new insight into the kinase evolution.植物通过跨膜类受体激酶（transmembrane receptor-like kinases, RLKs）感知周遭环境中的各类挑战并做出恰当响应，以此获得适应环境的能力。RLKs通过其可变的胞外结构域（extracellular domains, ECDs）识别多种配体，激活高度保守的胞内激酶结构域（intracellular kinase domains, KDs），并通过一套完善的下游信号级联反应（signaling cascade）调控多种不同的生物学功能。最新研究表明，油菜素甾醇不敏感1（brassinosteroid-insensitive 1, BRI1）家族与过剩小孢子母细胞1（excess microsporocytes 1, EMS1）——而非GASSHO1（GSO1）及其他RLKs——可通过同一信号通路调控不同的生物学功能，这引发了一个关键科学问题：以BRI1为代表的信号通路是如何实现功能特异性的？本研究首先证实，GSO1的激酶结构域无法在功能上替代BRI1的激酶结构域，因为嵌合型BRI1-GSO1无法挽救bri1突变体。随后我们鉴定出两个亚结构域S1与S2：若将BRI1的S1和S2替换为GSO1的对应序列，则该嵌合蛋白无法挽救bri1突变体；反之，若将嵌合型BRI1-GSO1的S1和S2替换为BRI1的对应序列，则可成功挽救bri1突变体，这表明S1与S2是赋予BRI1信号功能特异性的充分必要结构基础。进一步研究发现，尽管替换后的体外激酶活性存在差异，但BRI1激酶结构域内的其余所有亚结构域均可被GSO1的对应亚结构域在功能上替代，这提示其功能具有稳健性，且可通过多样化的激酶活性实现突变可塑性。有趣的是，S1包含作为变构热点（allosteric hotspot）的αC-β4环（αC-β4 loop），而S2则包含激酶激活环（kinase activation loop），二者据推测均可调控激酶活性。进一步分析显示，该特异性功能除依赖S1中的αC-β4环外，还需要β4与β5结构元件的参与。因此，我们认为BRI1通过这两个亚结构域的变构调控来确定其激酶功能特异性，进而调控其独特的生物学功能，这为激酶进化研究提供了全新的学术视角。