The structural basis of nucleic acid recognition by Toll-like receptors

描述（由申请人提供）：toll样受体（TLRs）在识别广泛保守的微生物细胞外结构时产生先天免疫信号反应。病毒RNA被识别！TLR3， TLR7和TLR8，微生物DNA由TLR9识别。直到最近，流行的范式是TLR9识别未甲基化的CpG DNA基序，这在细菌中大量存在，但在哺乳动物DNA中相对较少。然而，最近的研究包括我们自己的初步数据表明，TLR9与天然DNA配体的结合独立于它们的序列和甲基化状态。我们建议对允许TLR9识别微生物DNA的结构特性进行全面分析，包括序列、长度、双工含量、甲基化状态、主链化学（磷酸二酯与磷硫酸酯）、曲率和高阶结构（如结）。我们在初步研究中表明，DNA曲率诱导蛋白！HMGB1和组蛋白H2A、H2B显著增强了与TLR9 c端切割片段的结合，说明TLR9优先识别弯曲的DNA主干。为了确定DNA曲率对结合增强的影响程度，我们建议测量含有75至120个碱基对的DNA微环的TLR9结合亲和力。由于核小体可以诱导tlr依赖的自身免疫原性信号传导，我们建议对整个核小体作为TLR9配体进行体外生物物理分析！这些体外研究将在体内通过测量各种DNA或蛋白质-DNA配体（包括小环、核小体、连接和甲基化DNA配体）刺激的细胞中tlr9依赖性信号反应来验证。为了产生能够发出信号的受体，TLR7/8/9外结构域必须被蛋白水解切割。在第一项裂解TLR9的研究中，我们在初步数据中发现，N端和c端TLR9外畴片段都参与了配体结合和受体二聚化。因此，我们假设这两个片段在内体蛋白水解裂解后仍保持联系，并且裂解可能是TLR9经历配体诱导的激活受体的构象变化所必需的。为了验证这一假设，我们将探索两个TLR9外结构域片段之间的物理和功能关系，并利用生物物理学方法阐明蛋白水解激活的物理基础。TLR7/8/9结构信息的缺乏限制了我们对这些受体核酸识别的理解。我们建议使用电子冷冻显微镜和x射线晶体学作为补充方法来深入了解TLR9-DNA识别的结构基础。通过提供TLR9对微生物DNA识别的分子水平理解，该项目将提供必要的工具，以创造更有效的疫苗佐剂，以及一类具有广泛应用的新型抗炎治疗药物，特别是系统性红斑狼疮，哮喘，感染性休克综合征和器官移植排斥。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) generate an innate immune signaling response upon recognizing broadly conserved microbial extracellular structures. Viral RNA is recognized by!TLR3, TLR7 and TLR8, and microbial DNA is recognized by TLR9. Until recently the prevailing paradigm was that TLR9 recognized unmethylated CpG DNA motifs, which are abundant in bacteria but relatively scarce in mammalian DNA. However, recent studies including our own preliminary data suggest that TLR9 binds natural DNA ligands independently of their sequence and methylation state. We propose a comprehensive analysis of the structural properties that allow TLR9 to recognize microbial DNA including sequence, length, duplex content, methylation state, backbone chemistry (phosphodiester versus phosphorothioate), curvature and higher order structure (such as junctions). We show in preliminary studies that DNA curvature-inducing proteins!HMGB1 and histones H2A and H2B significantly enhance binding to the C-terminal cleavage fragment of TLR9, suggesting that TLR9 preferentially recognizes curved DNA backbones. To determine the extent to which DNA curvature alone is responsible for the binding enhancement, we propose to measure the TLR9 binding affinity of DNA minicircles containing 75 to 120 base pairs. Since nucleosomes can induce TLR-dependent auto immunogenic signaling, we propose an in vitro biophysical analysis of whole nucleosomes as TLR9 ligands! These in vitro studies will be validated in vivo by measuring TLR9-dependent signaling responses in cells stimulated with various DNA or protein-DNA ligands including minicircles, nucleosomes, junctions and methylated DNA ligands. The TLR7/8/9 ectodomains must be proteolytically cleaved in order to produce receptors that are capable of signaling. In the first study with cleaved TLR9, we show in our preliminary data that both the N- and C-terminal TLR9 ectodomain fragments participate in ligand binding and receptor dimerization. We therefore hypothesize that the two fragments remain associated after proteolytic cleavage in the endosome, and that cleavage may be necessary for TLR9 to undergo the ligand-induced conformational change that activates the receptor. To test this hypothesis, we will explore the physical and functional relationships between the two TLR9 ectodomain fragments, and elucidate the physical basis of proteolytic activation using biophysical approaches. The lack of structural information for TLR7/8/9 limits our understanding of nucleic acid recognition by these receptors. We propose to use electron cryomicroscopy and X-ray crystallography as complementary approaches to gain insight into the structural basis of TLR9-DNA recognition. By providing a molecular-level understanding of the recognition of microbial DNA by TLR9, this project will provide the necessary tools to create more potent vaccine adjuvants, and a new class of anti-inflammatory therapeutics with a wide range of applications including in particular systemic lupus erythematosus, asthma, septic shock syndrome and organ transplant rejection.