Interactions & mechanisms of function of the TAP complex

描述（由申请人提供）：与抗原加工复合物相关的转运蛋白包括三个亚基，TAP1， TAP2和tapasin。其中，TAP1和TAP2亚基属于ATP结合盒（ABC）跨膜转运蛋白家族，具有跨内质网（ER）膜转运肽的功能。Tapasin与两种TAP亚基相互作用，是增强TAP1/TAP2复合物结构稳定性所必需的。tapasin的er -腔结构域也与主要组织相容性复合体（MHC） I类分子相互作用，促进它们的组装。在本研究中，我们首先关注细胞质中的分子事件，并将研究TAP复合物在肽易位过程中TAP1和TAP2核苷酸结合域（NBD）的功能。我们研究了TAP NBD构象存在的证据，这些构象类似于DNA修复酶Rad50中NBD的相互作用。在这种相互作用中，每个TAP核苷酸结合位点将由一个NBD的Walker A基序和另一个NBD的特征基序的残基组成。利用TAP的病毒抑制剂、TAP底物以及在昆虫细胞中表达的适当设计的TAP1和TAP2突变体，我们将尝试重建TAP催化循环中发生的分子事件序列。在拟议研究的第二部分，重点是TAP复合物的er -腔面，特别是肽/MHC I类和tapasin/MHC I类相互作用的动力学。我们已经能够证明各种多肽缺陷MHC I类分子与tapasin之间的直接结合。在拟议的研究中，我们研究了tapasin功能的竞争位移模型，其中肽和tapasin竞争I类重链结合。我们将研究tapasin和MHC I类的哪些结构域对复杂的形成是重要的。我们还研究了MHC I类分子的tapasin依赖性与独立性的假设，这些假设是由I类分子的内在特性引起的。我们试图了解特定I类分子在内质网中增强TAP/tapasin结合的功能后果。综上所述，这些研究将深入了解肽如何转运到内质网，以及肽与MHC I类分子的组装如何在细胞内进行协调

DESCRIPTION (provided by applicant): The transporter associated with antigen processing complex comprises three subunits, TAP1, TAP2, and tapasin. Of these, the TAP1 and TAP2 subunits belong to the ATP binding cassette (ABC) family of transmembrane transporters, and function to translocate peptides across the endoplasmic reticulum (ER) membrane. Tapasin interacts with both TAP subunits, and is required for enhancing the structural stability of the TAP1/TAP2 complex. The ER-luminal domain of tapasin also interacts with major histocompatibility complex (MHC) class I molecules, facilitating their assembly. In the proposed studies, we first focus on the molecular events in the cytosol, and will investigate the functions of TAP1 and TAP2 nucleotide binding domains (NBD) during peptide translocation by TAP complexes. We examine evidence for the existence of conformations of the TAP NBD that resemble NBD interactions in the DNA repair enzyme Rad50. In such an interaction, each TAP nucleotide binding site would be comprised of residues from the Walker A motif of one NBD and the signature motif of the second NBD. Using viral inhibitors of TAP, TAP substrates, and appropriately designed TAP1 and TAP2 mutants expressed in insect cells, we will attempt to reconstruct the sequence of molecular events that occur during a TAP catalytic cycle. In the second part of the proposed studies, the focus is upon the ER-luminal face of TAP complexes, in particular on the dynamics of peptide/MHC class I and tapasin/MHC class I interactions. We have been able to demonstrate direct binding between various peptide-deficient MHC class I molecules and tapasin. In the proposed studies, we investigate a competitive displacement model for tapasin function, in which peptides and tapasin compete for class I heavy chain binding. We will examine which domains of tapasin and MHC class I are important for complex formation. We also examine the hypotheses that the tapasin-dependence vs. independence of MHC class I molecules results from intrinsic properties of the class I molecule. We seek to understand the functional consequences of enhanced TAP/tapasin binding in the ER by particular class I molecules. Taken together, these studies will provide insights into how peptide transport into the ER, and peptide assembly with MHC class I molecules, are orchestrated within the cell