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Clc sequence viewer 7.66/23/2023 However, the inhibition mechanism is still elusive and the EM model does not explain the biological function of the extended C-terminal region of ICP47 as to transport inhibition and its impact on the interaction with TAP. A recent cryo-electron microscopy study mapped the helix-loop-helix conformation of ICP47 in the central cavity formed by the two TAP subunits 25. This helix-loop-helix conformation has been confirmed at phospholipid membranes by solid-state NMR 24. It consists of two α-helices (residues 5-14 and 22-32) connected by a flexible linker. The structure of the active domain of ICP47 was determined by solution NMR 23. The active domain undergoes a large conformational change from random-coil in solution into an α-helical structure in a lipid environment 22. The active domain comprises residues 3-34 of ICP47, which displays the same inhibitory activity as the full-length protein 19, 21. For ICP47 binding, both TAP subunits are required 16, 17, but the TMD0s are dispensable 20. ICP47, also known as IE12, Vmw12, or IE5, binds to TAP from the cytosol ( K D = 50 nM) and arrests its function 15, 16, 17, 18, 19. Five out of the more than fifty ICPs are the immediate early polypeptides ICP0, 4, 22, 27, and 47 13, 14, which regulate the expression of other ICPs or help to evade the host’s immune system. After infection, cells start to synthesize infected cell polypeptides (ICPs). HSV infects mucosa and subsequently spreads via sensory neurons into ganglia, where it achieves a lifelong persistence 12. In herpes simplex viruses (HSV-1 and HSV-2), we find a distinct inhibition strategy, which also suppresses the MHC I surface presentation 11. For example, US6, the glycoprotein 6 of the cytomegalovirus unique short region, interacts with the ER-lumenal loops of TAP and prevents ATP binding at the cytosolic NBDs 6, 7, 8, 9, 10. Viruses evolved elaborate strategies to inhibit MHC I antigen processing by interfering with TAP function 5. The coreTAP complex is essential and sufficient for peptide binding and transport 3, while the TMD0s are necessary for assembly of the peptide loading complex. TMD and NBD form the coreTAP complex, which is connected by a short α-helix, named elbow helix, to the TMD0. The antigen translocation complex is composed of two half-transporters, TAP1 and TAP2 1, 2, which can both be divided into three functional modules: an N-terminal transmembrane domain (TMD0), the central transmembrane domain (TMD), and the cytosolic nucleotide-binding domain (NBD) 3, 4. After editing and ER quality control, stable peptide-MHC I complexes traffic to the cell surface in order to present their antigenic cargo to cytotoxic T-lymphocytes. As a centerpiece of the peptide loading complex, the heterodimeric ABC transporter TAP translocates proteasomal degradation products into the ER lumen, where they are loaded onto MHC I molecules. Within the cellular process of antigen presentation via major histocompatibility complex class I (MHC I) molecules, the transporter associated with antigen processing TAP is responsible for antigen compartmentalization. These new insights into the ICP47 inhibition mechanism can be applied for future structural analyses of the TAP complex. A per se destabilizing active domain inhibits the function of TAP, whereas a conserved C-terminal region additionally stabilizes the transporter. Based on our findings, we propose a dual interaction mechanism for ICP47. Binding of the active domain of ICP47 arrests TAP in an open inward facing conformation rendering the complex inaccessible for other viral factors. We unveiled a conserved region next to the active domain of ICP47 as essential for the complete stabilization of the TAP complex. ICP47-TAP fusion complexes are arrested in a stable conformation, as demonstrated by MHC I surface expression, melting temperature, and the mutual exclusion of herpesviral TAP inhibitors. These fusion complexes allowed us to determine the direction and positioning in the central cavity of TAP. Here, we report on a thermostable ICP47-TAP complex, generated by fusion of different ICP47 fragments. The herpesviral ICP47 inhibits TAP function, thereby suppressing an adaptive immune response. As a centerpiece of antigen processing, the ATP-binding cassette transporter associated with antigen processing (TAP) became a main target for viral immune evasion.
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