The interaction between outer membrane porins and toll-like receptors

描述(申请人提供)：在先天免疫系统中，Toll样受体(TLRs)对入侵的细菌、病毒、真菌和原生动物提供一线防御。耐人寻味的是，TLRs在没有已知成熟或选择的情况下就与它们的“非我”同源配体结合。一组TLR配体，外膜蛋白(OMPS)，或孔蛋白，是跨膜2桶蛋白。普遍识别一组物理性质差异很大的蛋白质的方法是很难想象的。我们假设TLRs最初基于静电吸引扫描外膜蛋白。我们进一步假设，一旦TLR被膜蛋白吸引，它们就会与主链结构元件结合，从而区分2-链和1-螺旋。这项为期两年的提案的目标是确定静电如何有助于TLR对OMP的识别。具体地说，我们计划：1.鉴定TLR2-PorB复合体的结构。我们已经用X射线结晶学确定了PorB的结构，这样PorB和TLR2现在都有可用的高分辨结构。我们进一步共提纯了该络合物，并进行了初步的电子显微镜成像，以显示确定共结构的可行性。2.研究静电对TLR2-PorB复合体亲和力的贡献。我们将使用盐和化学破坏来确定只有电荷效应是否有助于TLR2-络合物的亲和力。具体来说，我们将确定赖氨酸侧链的甲基化和乙酰化如何影响复杂的亲和力。3.确定在体外结合OMP的其他先天免疫受体的组合。虽然TLR2和PorB形成了一个信号复合体，但TLR2的其他组合对OMP的识别可能会导致不同的生理反应。我们克隆了5个天然免疫受体和3个OMP，以确定哪些受体和孔蛋白组合能够形成复合体。 公共卫生相关性：我们正在使用结构方法确定Toll样受体和外膜蛋白之间的识别机制。我们使用电子显微镜、核磁共振和结晶学的混合技术来研究这种在体内跨越两层膜的识别复合体。

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.