Neumann L, Tampé R
Philipps-Universität Marburg, Karl-von-Frisch-Str. 1, Marburg, 35033, Germany.
J Mol Biol. 1999 Dec 17;294(5):1203-13. doi: 10.1006/jmbi.1999.3329.
The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.
与抗原加工相关的转运体（TAP）在I类主要组织相容性复合体（MHC）介导的免疫监视中起关键作用。它将蛋白酶体复合物产生的肽转运到内质网（ER）中，以便加载到MHC I类分子上。在细胞表面，细胞毒性T淋巴细胞会监测这些MHC复合物的抗原负载情况。肽与TAP的结合是肽选择以及随后ATP依赖转运至内质网腔的关键步骤。为了研究TAP识别底物的途径，我们使用了用环境敏感荧光团标记的肽表位。与TAP结合后，检测到荧光底物的强烈荧光猝灭。这使我们能够通过均相分析实时分析TAP功能。肽-TAP复合物的形成由快速结合步骤和随后的转运复合物缓慢异构化组成。靠近荧光标记移动的质子供体基团会导致荧光猝灭。综上所述，这种肽诱导的结构重组可能反映了肽结合位点与TAP复合物内两个核苷酸结合域之间结构信息的相互作用。
