Molecular mechanism of TIRAP membrane targeting

描述(由申请人提供)：Toll样受体(TLRs)通过激活天然免疫反应为宿主防御提供了一种机制。激活的TLR[例如，通过细菌脂多糖(LPS)]二聚化，并通过其胞浆TIR结构域与接头蛋白相互作用，触发信号级联，最终导致参与促炎反应的蛋白表达。一种这样的接头蛋白是含有TIR结构域的适配器蛋白(TIRAP；也称为MAL)，它包含一个N端磷脂酰肌醇4，5-二磷酸(PtdIns(4，5)P2)结合区和一个C端TIR结构域，前者是质膜靶向所必需的，后者介导髓系分化初级反应基因88(MyD88)的结合。在与配体结合时，建议将内毒素结合蛋白TLR4招募到TIRAP所在的PtdIns(4，5)P2富集区。在这些位点，TIRAP通过TIR-TIR结构域的相互作用将MyD88招募到质膜上，从而将MyD88与激活的TLR4结合起来。PtdIns(4，5)P2介导的TIRAP募集被认为是TLR介导的信号转导所需的最早的细胞事件，因此，有人认为TIRAP定义了PtdIns(4，5)P2富含膜区的信号位点。因此，TIRAP如何在富含PtdIns(4，5)P2的膜上相互作用的细节对于了解该蛋白质如何在微生物检测时触发下游信号是至关重要的。在TIRAP的N端(氨基酸15-35)的一个保守的伸展已经被证明足以靶向质膜。我们的初步数据表明，这个区域，我们称之为PtdIns(4，5)P2结合基序(PBM)，在十二烷基磷胆碱胶束中折叠，并与PtdIns(4，5)P2结合。TIRAP PBM的胶束缔合和PtdIns(4，5)P2：胶束结合态的溶液结构将被求解和比较，以精确地绘制PtdIns(4，5)P2结合部位并确定多步膜插入的结构基础。TIRAP PBM与缺陷的PtdIns(4，5)P2结合突变体的相互作用动力学将通过核磁共振和表面等离子体共振检测来确定。TIRAP PBM的膜穿透深度和角度将通过顺磁自旋标记来阐明，以更好地了解结合如何影响膜的曲率。考虑到TIRAP与其他酸性磷脂的弱结合，这些分子在TIRAP膜插入中的作用将被确定。我们推测，TIRAP膜结合受PtdIns(4，5)P2头基团--三磷酸肌醇(Ins(1，4，5)P3)的调节，它在细胞外脂多糖存在的情况下积累。将测量TIRAP的InS(1，4，5)P3缔合动力学，并与PtdIns(4，5)P2的计算结果进行比较。因此，这些研究将为理解TIRAP膜靶向的机制和调控提供基础，可用于基于结构的设计高亲和力的特异性磷脂酰肌醇结合模块，最终有助于促炎反应。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) provide a mechanism for host defense by activating innate immune responses. Activated TLRs [e.g., by bacterial lipopolysaccharide (LPS)] dimerize, and interact with adaptor proteins through their cytosolic TIR domains to trigger a signaling cascade that ultimately leads to the expression of proteins involved in pro-inflammatory responses. One such adaptor protein is the TIR domain-containing adaptor protein (TIRAP; also known as MAL), which contains an N-terminal phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2)-binding region that is required for plasma membrane targeting and a C-terminal TIR domain, which mediates myeloid differentiation primary response gene 88 (MyD88) association. Upon ligand binding, the LPS-binding protein TLR4 is proposed to be recruited to PtdIns(4,5)P2-rich regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the plasma membrane via TIR-TIR domain interactions; thus TIRAP bridges MyD88 binding to activated TLR4. PtdIns(4,5)P2-mediated recruitment of TIRAP is considered to be the earliest cellular event required for TLR-mediated signaling and, consequently, it has been proposed that TIRAP defines the signaling sites at PtdIns(4,5)P2-rich membrane regions. Therefore, details of how TIRAP interacts in PtdIns(4,5)P2-rich membrane sites are crucial to understanding how the protein triggers downstream signaling upon microbial detection. A conserved stretch at the N-terminus of TIRAP (amino acids 15-35) has been shown to be sufficient to target the plasma membrane. Our preliminary data indicates that this region, which we name the PtdIns(4,5)P2 binding motif (PBM), folds in dodecylphosphocholine micelles, and binds PtdIns(4,5)P2. The solution structures of the micelle-associated and PtdIns(4,5)P2:micelle-bound states of TIRAP PBM will be solved and compared to precisely map the PtdIns(4,5)P2 binding site and define the structural basis of multi-step membrane insertion. Kinetics of the interactions of TIRAP PBM and deficient PtdIns(4,5)P2-binding mutants will be defined using NMR and surface plasmon resonance detection. The depth and angle of membrane penetration of TIRAP PBM will be elucidated with paramagnetic spin labels to better understand how binding influences membrane curvature. Given that TIRAP weakly binds to other acidic phospholipids, the contribution of these molecules in TIRAP's membrane insertion will be determined. We hypothesize that TIRAP membrane binding is regulated by the PtdIns(4,5)P2 head group, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which accumulates in the presence of extracellular LPS. The kinetics of TIRAP's Ins(1,4,5)P3 association will be measured and compared with those calculated for PtdIns(4,5)P2. Thus, these studies will provide a basis for understanding the mechanism and regulation of TIRAP's membrane targeting, which can be used for structure-based design of high affinity specific phosphoinositide-binding modules that ultimately contribute to pro-inflammatory responses.