Calcium regulatory proteins in plants; an NMR approach

钙在所有真核细胞中作为第二信使起着重要的作用。细胞内钙离子水平的升高可以通过与钙调素(CaM)等调节钙结合蛋白的结合而起到触发作用。总的来说，钙感受器蛋白可以改变细胞的生理状态。在植物中，细胞内钙离子水平升高是对各种非生物和生物胁迫的响应，如寒冷、干旱、盐、伤害或植物病原体的存在。我们最近报道了各种植物CaM及其类似蛋白结构域在载脂蛋白和钙离子饱和状态下的溶液结构。此外，我们还确定了CaM异构体与三种直接参与逆境反应的植物蛋白的复合体的三个高分辨结构：谷氨酸脱羧酶、MAPK-磷酸酶和液泡钙-ATPase。在所有三个案例中，都获得了以前从未见过的哺乳动物CaM复合体的新的复杂结构，突显了植物钙传感器蛋白家族出人意料的结构多样性。最近的基因组测序工作表明，所有的植物都含有大量的CaM、CaM样蛋白和CaM样蛋白结构域。例如，植物(和原生动物)含有一类独特的蛋白激酶(通常称为CDPK和CCaMK)，这是其他生物体中没有的。这些植物特有的酶含有它们自己的整合的CaM样结构域，这些结构域调节着它们的活性。我们计划解决一些最不同的调节域的三维结构，以找出它们与钙、它们的蛋白质靶标或其他钙感受器蛋白质相互作用的方式有何不同。此外，我们还将完成对一种高度分化的类CaM蛋白的研究，该蛋白在植物的盐胁迫反应中发挥作用。最后，我们将研究在各种植物中检测到的一种不同寻常的膜锚定版本的CaM。在这项研究计划中，我们将继续使用核磁共振波谱作为我们的主要-但不是唯一的-实验工具，以获得关于这些蛋白质及其在溶液状态下的复合体的详细结构和动态信息。

Calcium plays an important role as a secondary messenger in all eukaryotic cells. An increase in the cytosolic Ca2+ level can act like a trigger device by binding to regulatory calcium-binding proteins, such as calmodulin (CaM). Collectively the calcium-sensor proteins can redirect the physiological status of a cell. In plants the cytosolic Ca2+ level increases in response to various abiotic and biotic stresses such as cold, drought, salt, wounding or the presence of plant pathogens. We have recently reported the solution structures of various plant CaM's and CaM-like protein domains in their apo and Ca2+-saturated states. Moreover we have also determined three high-resolution structures of complexes of a CaM isoform with three plant proteins that are directly involved in mediating the stress response: glutamate decarboxylase, MAPK-phosphatase and a vacuolar calcium-ATPase. In all three cases novel complex structures were obtained, which had not been seen before for any mammalian CaM complexes, highlighting unexpected structural diversity of the plant calcium-sensor protein family. Recent genome sequencing efforts have revealed that all plants possess a large variety of CaM's, CaM-like proteins and CaM-like protein domains. For example, plants (and protozoa) contain a unique class of protein kinases (commonly known as the CDPK's and the CCaMK's) that are not found in other organisms. These plant-specific enzymes contain their own integrated CaM-like domains, which modulate their activity. We plan to solve the three-dimensional structures of some of the most distinct regulatory domains to find out how they differ in the way they interact with calcium, their protein targets, or other calcium-sensor proteins. Moreover we will complete our studies of a highly divergent CaM-like protein that plays a role in the salt stress response in plants. Finally we will study an unusual membrane anchored version of CaM that has been detected in various plants. For this research program we will continue to use Nuclear Magnetic Resonance (NMR) spectroscopy as our main -but not exclusive- experimental tool, to obtain detailed structural and dynamic information about these proteins and their complexes in the solution state.