Physical Basis of T Cell Activation by Superantigens

描述(由申请人提供)：超抗原(SAG)是一种 细菌或病毒来源，通过交联性T细胞刺激T细胞 受体(TCR)和主要组织相容性(MHC)II类分子。 刺激导致与大量释放相关的过激反应 致热和炎性细胞因子，通常紧随其后的是T细胞无能或 删除。已鉴定出两类SAGs：外源可溶性蛋白 由细菌分泌，如葡萄球菌肠毒素(SE)，以及 内源性逆转录病毒编码的跨膜蛋白，如小鼠的SAGs 乳腺肿瘤病毒(MTV SAG)。我们的目标是阐明其物理基础 细菌和病毒SAG对T细胞活化的影响 TCR-SAG和SAG-MHC络合物的X射线三维结构研究 结晶学技术并将这一信息与亲和力相关联 TCR-SAG和SAG-MHC相互作用的测量。 我们将确定具有代表性的TCR f3链的晶体结构-SAG和 SAG-MHC复合体，以定义SAG具有的不同策略 进化为结合TCR和MHC。我们之前确定了一个 小鼠TCR f3链与SEB络合。我们现在要确定的是 人TCR B链与中毒性休克综合征毒素-1(TSST-1)复合 链球菌致热外毒素C(SPEC)。为了鉴定高亲和力的锌离子 依赖于MHC II类分子上的SAG结合位点，我们最近解决了 与人类白细胞抗原-DR2a结合的SPEC携带髓鞘碱性蛋白(MBP)的自体多肽。 我们现在将把这项研究扩展到SEA和SED，它们交叉链接II类 APC上的分子。 SPEC-DR2a/MBP复合体的结构表明，SPEC使 与结合的MBP多肽接触，提示该多肽可以直接 影响SAG的绑定和呈现。人类白细胞抗原-DR2a的特异性亲和力 含有MBP多肽类似物的分子将被确定。的能力 向T细胞呈递SPEC的已定义的MHC/肽复合物将通过以下方式进行测试 用共价连接的单肽在表面表达DR分子 Il阴性的DAP-3细胞。我们还建议定义动力学和 控制细菌SAGs激活T细胞的亲和力参数 具有高亲和力和低亲和力的SPEC和TSST-1工程突变体 对TCR和MHC的毒性高于野生型毒素。 最后，为了建立病毒SAGs识别MHC的基础，我们 将表达可溶形式的MTV7 SAG用于直接结合和 与Ied类H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.