Molecular mechanism of TIRAP membrane targeting

描述（由申请人提供）：toll样受体（TLRs）通过激活先天免疫反应提供宿主防御机制。激活的tlr[例如，通过细菌脂多糖（LPS）]二聚化，并通过其细胞质TIR结构域与适配蛋白相互作用，触发信号级联，最终导致参与促炎反应的蛋白表达。其中一个这样的接头蛋白是含TIR结构域的接头蛋白（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，这是质膜靶向所必需的，还有一个c端TIR结构域，介导髓细胞分化初级反应基因88 （MyD88）关联。在配体结合后，lps结合蛋白TLR4被认为被募集到TIRAP所在的PtdIns（4,5）富含p2的区域。在这些位点上，TIRAP通过TIR-TIR结构域相互作用将MyD88招募到质膜上；因此TIRAP架起了MyD88与活化的TLR4结合的桥梁。PtdIns(4,5) p2介导的TIRAP募集被认为是tlr介导的信号传导所需的最早细胞事件，因此，有人提出TIRAP定义了PtdIns(4,5) p2富膜区域的信号传导位点。因此，TIRAP如何在PtdIns（4,5）富含p2的膜位点相互作用的细节对于理解蛋白质如何在微生物检测时触发下游信号至关重要。在TIRAP的n端（氨基酸15-35）有一个保守的拉伸，足以靶向质膜。我们的初步数据表明，这个区域，我们将其命名为PtdIns(4,5)P2结合基序（PBM），在十二烷基磷酸胆碱胶束中折叠，并结合PtdIns(4,5)P2。将对TIRAP PBM胶束结合态和PtdIns(4,5)P2的溶液结构进行求解和比较，以精确绘制PtdIns(4,5)P2结合位点，并确定多步插膜的结构基础。TIRAP PBM和缺乏PtdIns(4,5) p2结合突变体的相互作用动力学将使用核磁共振和表面等离子体共振检测来定义。用顺磁自旋标记来阐明TIRAP PBM的膜穿透深度和角度，以更好地了解结合如何影响膜曲率。考虑到TIRAP与其他酸性磷脂的弱结合，这些分子在TIRAP的膜插入中的作用将被确定。我们假设TIRAP膜结合是由PtdIns(4,5)P2头基团，肌醇1,4,5-三磷酸（Ins(1,4,5)P3）调节的，它在细胞外LPS存在下积累。将测量TIRAP的Ins(1,4,5)P3关联动力学，并与PtdIns(4,5)P2计算的动力学进行比较。因此，这些研究将为了解TIRAP的膜靶向机制和调控提供基础，可用于基于结构的设计高亲和力特异性磷酸肌苷结合模块，最终促进促炎反应。

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.