Physical Basis of T Cell Activation by Superantigens

描述（由申请人提供）：超抗原（SAG）是 通过交联T细胞刺激T细胞的细菌或病毒来源 受体（TCR）和主要组织相容性（MHC）II类分子。 刺激导致过度活跃的反应与大量释放 致热性和炎性细胞因子，通常随后是T细胞无反应性或 删除。已经鉴定了两类SAG：外源可溶性蛋白 由细菌分泌，如葡萄球菌肠毒素（SE），和 内源性逆转录病毒编码的跨膜蛋白，如小鼠的SAG 乳腺肿瘤病毒（Mtv SAG）。我们的目标是阐明 通过测定细菌和病毒SAG对T细胞活化的影响， X射线测定TCR-SAG和SAG-MHC复合物的三维结构 晶体学技术，并将这些信息与亲和力 TCR-SAG和SAG-MHC相互作用的测量。 我们将确定代表性TCR f3链-SAG的晶体结构， SAG-MHC复合物，以确定SAG具有的不同策略 进化为结合TCR和MHC。我们先前确定了一种 与SEB复合的小鼠TCR f3链。我们现在将确定 与中毒性休克综合征毒素-1（TSST-1）复合的人TCR B链，和 链球菌致热原外毒素C（SPEC）。为了鉴定高亲和性，Zn 2 + 依赖于SAG结合位点的MHC II类，我们最近解决了结构， SPEC与HLA-DR 2a结合，该HLA-DR 2a具有来自髓鞘碱性蛋白（MBP）的自身肽。 我们现在将这项研究扩展到SEA和SED，它们将II类药物交联 APC分子。 SPEC-DR 2a/MBP复合物的结构表明，SPEC使广泛的 与结合的MBP肽接触，表明肽可以直接 影响SAG结合和呈递。SPEC与HLA-DR 2a的亲和力 将测定带有MBP肽类似物的分子。的能力 将通过以下方法检测将SPEC呈递给T细胞的确定的MHC/肽复合物： 在表面上表达具有共价连接的单肽的DR分子， II类阴性DAP-3细胞。我们还建议定义动力学和 通过细菌SAG控制T细胞活化的亲和力参数， 具有较高和较低亲和力的SPEC和TSST-1的工程突变体 比野生型毒素的TCR和MHC。 最后，为了建立病毒SAGs识别MHC的基础，我们 将表达用于直接结合的Mtv 7 SAG的可溶形式， 与I艾德H类分子的共结晶实验。

DESCRIPTION (provided by applicant): Superantigens (SAGs) are proteins of bacterial or viral origins that stimulate T cells by cross-linking T cell receptors (TCRs) and major histocompatibility (MHC) class II molecules. Stimulation leads to hyperactive responses associated with the massive release of pyrogenic and inflammatory cytokines, usually followed by T cell anergy or deletion. Two classes of SAGs have been identified: exogenous soluble proteins secreted by bacteria, such as the staphylococcal enterotoxins (SEs), and endogenous retroviral-encoded transmembrane proteins, such as the SAGs of mouse mammary tumor viruses (Mtv SAGs). Our goal is to elucidate the physical basis of T cell activation by bacterial and viral SAGs through determination of the three-dimensional structure of TCR-SAG and SAG-MHC complexes by X-ray crystallographic techniques and to correlate this information with affinity measurements of TCR-SAG and SAG-MHC interactions. We will determine the crystal structures of representative TCR f3 chain-SAG and SAG-MHC complexes in order to define the diverse strategies that SAGs have evolved for binding TCR and MHC. We previously determined the structures of a mouse TCR f3 chain complexed with SEB. We will now determine the structures of a human TCR B chain complexed with toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxin C (SPEC). To identify the high-affinity, Zn2+ dependent SAG binding site on MHC class II, we recently solved the structure of SPEC bound to HLA-DR2a bearing a self-peptide from myelin basic protein (MBP). We will now extend this study to SEA and SED, which cross-link class II molecules on APCs. The structure of the SPEC-DR2a/MBP complex reveals that SPEC makes extensive contacts with the bound MBP peptide, suggesting that peptide can directly influence SAG binding and presentation. The affinity of SPEC for HLA-DR2a molecules bearing analogs of the MBP peptide will be determined. The ability of defined MHC/peptide complexes to present SPEC to T cells will be tested by expressing DR molecules with covalently-linked single peptides on the surface of class Il-negative DAP-3 cells. We also propose to define the kinetic and affinity parameters governing T cell activation by bacterial SAGs by engineering mutants of SPEC and TSST-1 with both higher and lower affinities for TCR and MHC than the wild type toxins. Finally, in order to establish the basis for MHC recognition by viral SAGs, we will express soluble forms of Mtv7 SAG for use in direct binding and co-crystallization experiments with the I Ed class H molecule.