Interactions & mechanisms of function of the TAP complex

描述(申请人提供)：与抗原处理复合体相关的转运蛋白由三个亚基组成：TAP1、TAP2和Tapasin。其中，TAP1和TAP2亚基属于三磷酸腺苷结合盒(ABC)跨膜转运蛋白家族，具有跨内质网(ER)膜转运多肽的功能。Tapasin与两个TAP亚基相互作用，是增强TAP1/TAP2复合体结构稳定性所必需的。Tapasin的内质网结构域还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。在拟议的研究中，我们首先关注胞浆中的分子事件，并将研究TAP1和TAP2核苷酸结合域(NBD)在TAP复合体多肽易位过程中的功能。我们检查了在DNA修复酶Rad50中存在类似于NBD相互作用的TAP NBD构象的证据。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基序和第二个NBD的签名基序的残基组成。使用TAP、TAP底物的病毒抑制剂，以及在昆虫细胞中表达的适当设计的TAP1和TAP2突变体，我们将试图重建在TAP催化循环中发生的分子事件序列。在拟议的研究的第二部分，重点放在TAP复合体的内质网-腔面，特别是多肽/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缺乏多肽的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功能的竞争置换模型，在该模型中，多肽和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结构的形成是重要的。我们还检验了假设，即MHC I类分子的Tapasin依赖性与独立性是由I类分子的内在性质造成的。我们试图了解特定的I类分子在内质网中增强TAP/TAPASIN结合的功能后果。综上所述，这些研究将为多肽进入内质网的运输以及与MHC I类分子的多肽组装如何在细胞内协调提供深入的见解

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