Molecular Genetic Analysis Of Lymphocyte Function

如上所述，第(1)和(2)部分涉及本项目的MHC-I方面，通常旨在了解MHC-I分子负载自身或抗原肽的分子细节。虽然已经确定了数百个MHC-I和MHC-I类三维结构，但这些结构都不是该分子的肽受体(PR)形式。在以前的研究中，我们确定了代表部分MHC-I分子H2-LD的多肽表位的三维结构，该表位仅暴露在部分未折叠的多肽受体(PR)MHC-I分子上。这随后定义了分子PR形式的一个方面，该形式被用作完全水化模型的分子动力学模拟的输入。这种动力学模拟得到了广泛的检验，并提供了对MHC-I分子从亚稳态PR形式转变为稳定PL形式的结构理解。对这两种结构以及从PR到PL的转变的分析表明了MHC-I分子如何工作的详细机制。为了扩大我们对多肽负载性质的理解，我们设计了参与MHC-I负载的主要伴侣蛋白，Tapasin和Erp57。此外，我们还设计了一种类似TAPASIN的分子，称为TAPBPR，它的氨基酸序列与TAPASIN大约20%相同，并进行了研究，检测其与MHC-I的PR形式结合的性质。这些研究表明，TAPBPR与一种无肽的、可接受多肽的MHC-I相互作用，并且这种相互作用在多肽结合时松弛。对TAPBPR/MHC-I相互作用的进一步研究表明，抗原肽与MHC-I直接相互作用，而不是TAPBPR组分。此外，多肽与MHC-I分子的相互作用与多肽与MHC-I分子的结合强度(亲和力)定量相关。在对MHC-I/肽相互作用的进一步研究中，我们探索了抗逆转录病毒药物阿巴卡韦在与MHC-I结合和扭曲MHC-I易感等位基因结合的自肽谱中所起的作用。特别是，我们通过对易感MHC-I等位基因--人类白细胞抗原-B*57：01结合的多肽的肽序列分析表明，在阿巴卡韦存在和不存在的情况下，该药物可以改变B*57：01结合的多肽。这为在接受药物治疗的高比例的人类白细胞抗原-B*57：01个体中观察到的严重过敏反应提供了解释。我们已经建立了表达不同形式的人类白细胞抗原-B*57：01的转基因小鼠系，作为药物引起急性超敏反应的动物模型。这些转基因动物可以免疫产生针对人类白细胞抗原B*57：01的CD8-T细胞反应。 该项目的第三部分集中于T细胞受体识别抗原的结构和功能研究，这如何导致T细胞信号，以及这如何导致自身免疫性疾病。为了为了解TCR中抗原特异性的结构变化提供基线，我们确定了病毒特异性的MHC-I限制性TCR的X射线结构，以及它与MHC-I/病毒抗原配体的复合体。值得注意的是，尽管MHC/肽复合体具有相对刚性的结构，但TCR显示其CDR3α和β环有很大的移动，这表明配体结合的飞投机制。这个实验室的其他项目也反映了这种飞掷机制的进一步特征。通过新的生物物理技术探索TCR/MHC-I相互作用的其他研究正在进行中。特别是，我们在探索TCR/MHC相互作用的二维亲和力测量的研究中进行了合作。此外，我们还合作探索了使用核磁共振来研究TCR的构象变化，这些变化伴随着高亲和力多肽/MHC复合体的结合。这些研究为T细胞活化可能的变构机制提供了新的见解。 了解TCR介导的自身免疫方面的其他方法包括：1)自身免疫T细胞在自身免疫性胃炎转基因小鼠模型中识别的抗原肽的特征；以及2)与其抗原肽结合的MHC-II分子的结构测定。我们测定了IAD与Th2多肽(PLL)的高分辨X射线结构，以及另外两个与PLL相关但具有较高内在亲和力的IAD/多肽复合体的结构。在高分辨率下测定的这些结构揭示了IAD的一个以前未被识别的结合基序(利用多肽的1、4、6、7和9残基)，特别是关于多肽第9位的谷氨酸偏好。这为理解自身免疫TCR与自身MHC-II/肽复合体的相互作用提供了一个框架。这些实验结构研究允许对另一种被称为PIT的与IAD结合的胃炎诱导肽进行建模，并进一步深入了解自身免疫性胃炎的分子基础。相关锚点的这一主题和与MHC结合的多肽的拓扑结构与其他MHC-II/自身抗原复合体的相似性是一致的，并提示了一些可能与自身免疫抗原特异相关的共同特征。

Parts (1) and (2) as listed above, deal with the MHC-I aspects of this project, and in general are directed to understand the molecular details of the loading of MHC-I molecules with self or antigenic peptides. Although hundreds of MHC-I and MHC-I-like three-dimensional structures have been determined, none of these is of a peptide-receptive (PR) form of the molecule. In previous studies, we determined the three-dimensional structure of a peptide epitope representative of a portion of the MHC-I molecule H2-Ld that is exposed only on partially unfolded, peptide receptive (PR) MHC-I molecules. This then defined one aspect of the PR form of the molecule, which was used as the input for molecular dynamics simulations of a fully hydrated model. This dynamics simulation has been examined extensively and provides a structural understanding of the way that MHC-I molecules change their shape from the metastable PR form to their stable PL form. Analysis of the two structures and of the transition from PR to PL suggests a detailed mechanism of how the MHC-I molecule works. To extend our understanding of the nature of peptide-loading, we have engineered the main chaperones involved in MHC-I loading, tapasin and Erp57. In addition, we have engineered a tapasin like molecule, known as TAPBPR, which is about 20% identical in amino acid sequence to tapasin and have undertaken studies examining the nature of its binding to the PR form of MHC-I. These studies suggest that TAPBPR interacts with a peptide-free, peptide-receptive form of MHC-I, and that this interaction is relaxed upon peptide binding. Additional studies of the TAPBPR/MHC-I interaction reveal direct interaction of antigenic peptides with the MHC-I and not the TAPBPR component of the complex. Furthermore, the interaction of peptide with the MHC-I molecule is quantitatively related to the strength of binding (the affinity) of the peptide for the MHC-I molecule. In additional studies of MHC-I/peptide interactions, we have explored the role that the anti-retroviral drug, abacavir, plays in binding to MHC-I and distorting the self-peptide repertoire bound by susceptible MHC-I alleles. In particular, we have shown, by peptide sequence analysis of the self peptides bound to the susceptible MHC-I allele, HLA-B*57:01, in the presence and absence of abacavir, that this drug can change the peptides that B*57:01 binds. This provides an explanation for the severe hypersensitivity reactions that are observed in a high proportion of HLA-B*57:01 individuals who receive the drug. We have developed transgenic mouse lines expressing various forms of HLA-B*57:01 as animal models for the effects of drugs in causing acute hypersensitivity reactions. These transgenic animals can be immunized to generate HLA-B*57:01-specific CD8-T cell responses. The third part of this project is focused on structural and functional studies of T cell receptor recognition of antigens, how this leads to T cell signaling, and how this leads to autoimmune disease. To provide a baseline for understanding antigen-specific structural changes in the TCR, we have determined the X-ray structure of a viral specific, MHC-I-restricted TCR, as well as its complex with its MHC-I/viral antigen ligand. Remarkably, although the MHC/peptide complex has a relatively rigid structure, the TCR shows great movement of its CDR3 alpha and beta loops, indicative of a fly-casting mechanism for ligand engagement. Further characterization of this fly-casting mechanism is reflected in other projects from this laboratory. Additional studies are underway to explore TCR/MHC-I interactions by novel biophysical techniques. In particular we have collaborated in studies that explore measurements of two-dimensional affinities of TCR/MHC interactions. In addition, we have collaboratively explored the use of NMR to study conformational changes in the TCR that accompany binding of a high affinity peptide/MHC complex. These studies offer new insight into possible allosteric mechanisms that contribute to T cell activation. Other approaches to understanding the TCR mediated aspects of autoimmunity include: 1) the characterization of antigenic peptides recognized by the autoimmune T cells in transgenic mouse models of autoimmune gastritis; and 2) the structural determination of MHC-II molecules bound to their antigenic peptides. We have determined the high resolution X-ray structure of IAd in complex with the Th2 peptide known as PLL, as well as the structures of two other IAd/peptide complexes in which the peptides are related to PLL, but are of higher intrinsic affinity. These structures, determined at high resolution reveal a previously unrecognized binding motif (exploiting residues 1,4,6,7, and 9 of the peptide) for IAd, particularly with respect to the preference of glutamic acid at position 9 of the peptide. This provides a framework for understanding interactions of autoimmune TCR with self MHC-II/peptide complexes. These experimental structural studies permitted the modeling of another gastritis-inducing peptide, known as PIT, bound to IAd, and provide further insight into the molecular basis of autoimmune gastritis. Similarities in this theme of the relevant anchors and the topology of the peptide bound to the MHC are consistent with other MHC-II/autoantigen complexes and suggest some common features that may be specifically relevant to autoimmune antigens.