Molecular mechanism of TIRAP membrane targeting

TIRAP膜靶向的分子机制

• 批准号: 8700070
• 负责人: Daniel Guillermo Capelluto
• 金额: $ 8.05万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700070
• 关键词: Adaptor Signaling Protein; Affinity; Amino Acid Motifs; Amino Acids; Binding; Binding Sites; Biological Assay; C-terminal; CD14 Antigen; Cell membrane; Chimera organism; Complex; Data; Detection; Dimerization; Dissociation; Environment; Event; Exhibits; Gene Expression; Genes; Head; Host Defense Mechanism; Immune response; Inflammatory; Inflammatory Response; Inositol; Interleukin-1 Receptors; Kinetics; Ligand Binding; Lipid Bilayers; Lipid Binding; Lipids; Lipopolysaccharides; Maps; Measures; Mediating; Membrane; Membrane Lipids; Micelles; Molecular; Molecular Structure; Mutate; Myelogenous; N-terminal; NMR Spectroscopy; Names; Natural Immunity; Nature; Nucleic Acid Regulatory Sequences; Penetration; Phosphatidylinositols; Phospholipids; Physiological; Production; Property; Proteins; Recruitment Activity; Regulation; Reporting; Role; Signal Transduction; Signaling Protein; Site; Solutions; Sorting - Cell Movement; Specificity; Spin Labels; Stretching; Structure; Surface Plasmon Resonance; TLR4 gene; Tail; Titrations; Toll-like receptors; base; design; dodecylphosphocholine; extracellular; lipopolysaccharide-binding protein; microbial; mutant; pathogen; phosphatidylinositol phosphate, PtdIns(4,5)P2; protein complex; protein expression; public health relevance; receptor-mediated signaling; research study; response; three dimensional structure; transcription factor

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.

描述（由申请人提供）：Toll 样受体（TLR）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，并通过其胞质 TIR 结构域与接头蛋白相互作用，触发信号级联反应，最终导致参与促炎反应的蛋白质表达。其中一种接头蛋白是含 TIR 结构域的接头蛋白 (TIRAP；也称为 MAL)，它包含质膜靶向所需的 N 端磷脂酰肌醇 4,5-二磷酸 (PtdIns(4,5)P2) 结合区和介导骨髓分化初级反应基因 88 的 C 端 TIR 结构域 （MyD88）协会。配体结合后，LPS 结合蛋白 TLR4 被募集到 TIRAP 所在的富含 PtdIns(4,5)P2 的区域。在这些位点，TIRAP 通过 TIR-TIR 结构域相互作用将 MyD88 募集至质膜；因此，TIRAP 将 MyD88 与激活的 TLR4 结合桥接起来。 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的细胞事件，因此，有人提出 TIRAP 定义了富含 PtdIns(4,5)P2 的膜区域的信号传导位点。因此，TIRAP 如何在富含 PtdIns(4,5)P2 的膜位点中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传导至关重要。 TIRAP N 末端的保守延伸（氨基酸 15-35）已被证明足以靶向质膜。我们的初步数据表明，我们将这个区域命名为 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。将求解并比较 TIRAP PBM 的胶束相关态和 PtdIns(4,5)P2:胶束结合态的溶液结构，以精确绘制 PtdIns(4,5)P2 结合位点并定义多步膜插入的结构基础。 TIRAP PBM 和缺陷 PtdIns(4,5)P2 结合突变体相互作用的动力学将使用 NMR 和表面等离子共振检测来定义。 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曲率。鉴于 TIRAP 与其他酸性磷脂的结合较弱，因此将确定这些分子在 TIRAP 膜插入中的贡献。我们假设 TIRAP 膜结合受到 PtdIns(4,5)P2 头基、肌醇 1,4,5-三磷酸 (Ins(1,4,5)P3) 的调节，其在细胞外 LPS 存在时积累。将测量 TIRAP 的 Ins(1,4,5)P3 关联的动力学，并与 PtdIns(4,5)P2 计算的动力学进行比较。因此，这些研究将为理解 TIRAP 膜靶向的机制和调节提供基础，可用于基于结构的高亲和力特异性磷酸肌醇结合模块的设计，最终有助于促炎反应。