Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1.
Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1.
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DOI：
10.7554/elife.00813
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发表时间：
2013-07-30
期刊：
影响因子：
7.7
通讯作者：
Kuriyan J
中科院分区：
文献类型：
--
作者：
Iwig JS;Vercoulen Y;Das R;Barros T;Limnander A;Che Y;Pelton JG;Wemmer DE;Roose JP;Kuriyan J
RasGRP 1和SOS是Ras特异性核苷酸交换因子，在淋巴细胞发育中具有不同的作用。RasGRP 1在某些癌症和自身免疫性疾病中很重要，但与SOS相反，其调控机制知之甚少。激活信号导致RasGRP 1和Ras参与的膜募集，但目前尚不清楚RasGRP 1和Ras之间的相互作用如何在缺乏此类信号的情况下被抑制。我们提出了一个晶体结构的片段RasGRP 1中的Ras结合位点被阻断的域间连接器和膜相互作用表面的RasGRP 1是隐藏在一个二聚化界面，可以稳定的C-末端寡聚化结构域。NMR数据表明，钙结合到监管模块产生大量的构象变化，是不兼容的非活性组件。这些特征使RasGRP 1保持在非活性状态，准备通过钙和膜定位信号激活。DOI：http://dx.doi.org/10.7554/eLife.00813.001人体内的单个细胞必须生长、分裂或专门化以执行所需的任务。这些细胞的命运通常由Ras家族中的蛋白质指导，Ras家族检测来自身体其他部位的信号并协调每个细胞内的反应。这些蛋白质的活性必须严格控制，因为如果Ras蛋白质没有得到适当的调节，可能会导致癌症和发育疾病。与小分子GTP的结合激活Ras并引起构象变化，使其能够与细胞中各种信号通路中的其他蛋白质相互作用。GTP被称为核苷酸交换因子的蛋白质装载到Ras中，这些蛋白质可以用“新鲜”的核苷酸替换“使用过的”核苷酸以激活Ras。这些核苷酸交换因子也受到严格调控。例如，许多交换因子的基因仅在接收到特定信号后才被打开，这可以将它们的存在限制在限定的时间和位置（例如，细胞或组织）。此外，当激活信号不存在时，核苷酸交换因子通常驻留在细胞质中，而Ras蛋白保持与细胞内的脂质膜结合。RasGRP 1是一种控制免疫细胞发育的核苷酸交换因子，如果调节不当，可能导致白血病和狼疮。然而，关于RasGRP 1的许多问题仍然没有答案，包括它如何能够保持不活动，以及它如何被各种不同的信号激活。Iwig等人现在已经揭示了RasGRP 1抑制免疫细胞中Ras信号传导的机制，通过解决RasGRP 1的两个片段的结构，然后使用结构，生物化学和基于细胞的方法来探索它是如何被激活的。这些分析表明，无活性的RasGRP 1采用的构象，其中一个调控元件阻断Ras结合位点的访问。令人惊讶的是，RasGRP 1可以形成二聚体;这隐藏了与膜相关的蛋白质部分，从而使RasGRP 1远离Ras。Iwig等人还发现，两种信号，钙离子和一种称为二酰基甘油的脂质，通过改变RasGRP 1的构象并将其招募到膜上来克服这些抑制机制。这些研究为理解RasGRP 1中的疾病相关突变如何绕过确保适当免疫细胞发育的调节机制提供了一个框架，并且对于开发抑制RasGRP 1活性的治疗药物至关重要。DOI：http://dx.doi.org/10.7554/eLife.00813.002网站
RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 Individual cells within the human body must grow, divide or specialize to perform the tasks required of them. The fates of these cells are often directed by proteins in the Ras family, which detect signals from elsewhere in the body and orchestrate responses within each cell. The activities of these proteins must be tightly controlled, because cancers and developmental diseases can result if Ras proteins are not properly regulated. Binding to the small molecule GTP activates Ras and causes conformational changes that allow it to interact with other proteins in various signaling pathways in the cell. GTP is loaded into Ras by proteins called nucleotide exchange factors, which can replace ‘used’ nucleotides with ‘fresh’ ones to activate Ras. These nucleotide exchange factors are also tightly regulated. For example, the genes for many exchange factors are only switched on after particular signals are received, which can restrict their presence to defined times and locations (e.g., cells or tissues). Also, when activating signals are absent, nucleotide exchange factors commonly reside in the cytoplasm, whereas the Ras proteins remain bound to lipid membranes inside the cell. RasGRP1 is a nucleotide exchange factor that controls the development of immune cells, and leukemia and lupus can result if it is not regulated correctly. However, many questions about RasGRP1 remain unanswered, including how it is able to remain inactive, and how it is activated by various different signals. Iwig et al. have now revealed the mechanisms through which RasGRP1 suppresses Ras signaling in immune cells by solving the structures of two fragments of RasGRP1 and then using a combination of structural, biochemical and cell-based methods to explore how it is activated. These analyses revealed that inactive RasGRP1 adopts a conformation in which one of its regulatory elements blocks access to the Ras binding site. Surprisingly, RasGRP1 can form dimers; this hides the portions of the protein that associate with the membrane and thereby keeps RasGRP1 away from Ras. Iwig et al. also found that two signals, calcium ions and a lipid called diacylglycerol, overcome these inhibitory mechanisms by changing the conformation of RasGRP1 and recruiting it to the membrane. These studies provide a framework for understanding how disease-associated mutations in RasGRP1 bypass the regulatory mechanisms that insure proper immune cell development, and will be critical for developing therapeutic agents that inhibit RasGRP1 activity. DOI: http://dx.doi.org/10.7554/eLife.00813.002