The interaction between outer membrane porins and toll-like receptors

描述（由申请人提供）：在先天免疫系统中，toll样受体（tlr）提供抵抗入侵细菌、病毒、真菌和原生动物的一线防御。有趣的是，tlr结合它们的“非自体”同源配体没有已知的成熟或选择。一组TLR配体，外膜蛋白（OMPs）或孔蛋白，是跨膜的2桶蛋白。对一组在物理性质上有很大变化的蛋白质进行普遍识别的方法是难以想象的。我们假设tlr最初基于静电吸引力扫描外膜蛋白。我们进一步假设，一旦tlr被膜蛋白吸引，它们就会结合到主链结构元件上，从而区分出2链和1螺旋。这项为期2年的提案的目标是确定静电如何促进tlr对omp的识别。具体来说，我们计划：1。鉴定TLR2-PorB复合物的结构。我们已经通过x射线晶体学确定了PorB的结构，这样PorB和TLR2现在都有了可用的高分辨率结构。我们进一步对该配合物进行了共纯化，并采取了初步的电子显微镜成像，以证明确定共结构的可行性。2. 研究静电对TLR2-PorB复合物亲和力的影响。我们将使用盐和化学破坏来确定是否仅带电效应有助于tlr2复合物的亲和力。具体来说，我们将确定甲基化和乙酰化赖氨酸侧链如何影响复杂的亲和力。3. 鉴定体外结合omp的其他先天免疫受体组合。虽然TLR2和PorB形成一个信号复合物，但其他tlr组合对omp的识别可能导致不同的生理反应。我们克隆了5个先天免疫受体和3个omp，以确定哪些受体和孔蛋白的组合能够形成复合物。

DESCRIPTION (provided by applicant): In the innate immune system, Toll-like receptors (TLRs) provide a front-line defense against invading bacteria, viruses, fungi and protozoa. Intriguingly, TLRs bind their "non-self" cognate ligand without known maturation or selection. One set of TLR ligands, the Outer Membrane Proteins (OMPs), or porins, are transmembrane 2-barrel proteins. The method of universal recognition of a group of proteins that have large variability in their physical properties is difficult to envision. We hypothesize that TLRs initially scan outer membrane proteins based on electrostatic attraction. We further hypothesize that once TLRs are attracted to a membrane protein, they bind to main chain structural elements thus differentiating 2-strands from 1-helices. The goal of this 2-year proposal is to identify how electrostatics contribute to the recognition of OMPs by TLRs. Specifically, we plan to: 1. Identify the structure of the TLR2-PorB complex. We have already determined the structure of PorB by x-ray crystallography, such that both PorB and TLR2 now have available high-resolution structures. We have further co-purified the complex and taken initial electron microscopy imagers to show feasibility of determination of a co-structure. 2. Investigate the contributions of electrostatics to the affinity of the TLR2-PorB complex. We will use salt and chemical disruption to identify if charge-only effects contribute to the affinity of the TLR2-complex. Specifically, we will identify how methylation and acetylation of lysine side chains affects complex affinity. 3. Identify additional combinations of innate immunity receptors that bind OMPs in vitro. While TLR2 and PorB form one signaling complex, recognition of OMPs by other combinations of TLRs may result in different physiological responses. We have cloned 5 innate immunity receptors and 3 OMPs to identify which combinations of receptors and porins are capable of forming a complex. PUBLIC HEALTH RELEVANCE: We are working to define the mechanisms of recognition between toll-like receptors and outer membrane proteins using a structural approach. We use a hybrid of electron microscopy, NMR, and crystallography to investigate this recognition complex, which spans two membranes in vivo.