Structural insights into substrate binding to the ABC transport complex TAP
Zusammenfassung der Projektergebnisse
The heterodimeric ABC transporter associated with antigen processing (TAP) displays an essential factor in the adaptive immune response by transporting proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum for loading of MHC class I molecules. Peptide bound MHC class I are presented on the plasma membrane to cytotoxic T-cells for inspection to erase viral infected or malignant transformed cells. Several viruses have developed strategies to escape this surveillance by interfering with TAP function. Therefore, the objective of this project was to elucidate structural and mechanistic aspects of peptide binding and transport of TAP. A breakthrough in the beginning of the project was the establishment of the fermenter-based expression of TAP in Pichia pastoris, yielding 30 mg of TAP per liter cell culture, in combination with the functional purification and reconstitution, first in liposomes and later in nanodiscs. By a detailed cysteine-scanning and cross-linking approach, we illuminated the transmission interface between the transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) of TAP1 and TAP2 and assigned different functions for peptide binding and transport to cytosolic loops in the interface. Additionally, we studied the communication between the two cytosolic NBDs, which dimerize during the transport cycle to hydrolyze ATP to power peptide transport. The core of the interface between both NBDs is formed by the highly conserved D-loop. Substitution of the aspartate by alanine of the D-loop in TAP1 interrupted ATPase activity of TAP and rendered the mutant in a nucleotide-gated peptide facilitator, which could no longer translocate the peptides against a gradient. Remarkably, peptide accumulation on the ER- lumenal side did not exceed 16 µM reflecting a trans-inhibition mechanism in which transported peptide is bound to the low affinity peptide binding side and inhibits ATP hydrolysis and therefore progression of the transport cycle. By fluorescence correlation spectroscopy we demonstrated that only one peptide binds to TAP. Interestingly, cysteine 213 of TAP2, distant from the peptide binding pocket, regulates substrate specificity since replacement of C213 changed peptide specificity. By continuous wave and pulsed electron paramagnetic resonance spectroscopy, the conformation of the peptide in the peptide binding site was determined. Independent of the length of the peptide, the terminal residues are separated by 2.5 nm, which is in perfect agreement with the length specificity of TAP. Longer peptides engage a kinked conformation to fit in. By dynamic nuclear polarization enhanced magic angle spinning solidstate nuclear magnetic resonance spectroscopy, we could proof that the applied 9-mer peptide binds in an extended conformation to TAP. Docking the peptide structure into a structural model of human TAP derived from TAP-related heterodimeric ABC transport complex TmrAB allowed the prediction of the binding pocket, which is split in two halves for the N- and C-terminal residues. Finally, by single particle cryo-EM and by X-ray crystallography we solved the structure of the functional bacterial TAP homologue TmrAB in the apo state where the TMDs adopt an inward open conformation.
Projektbezogene Publikationen (Auswahl)
- (2008) Peptide specificity and lipid activation of the lysosomal transport complex ABCB9 (TAPL). J Biol Chem 283, 17083–17091
Zhao, C., Haase, W., Tampé, R., and Abele, R.
(Siehe online unter https://doi.org/10.1074/jbc.m801794200) - (2009) Peptide trafficking and translocation across membranes in cellular signaling and self-defense strategies. Curr Opin Cell Biol 21, 508–515
Abele, R., and Tampé, R.
(Siehe online unter https://doi.org/10.1016/j.ceb.2009.04.008) - (2009) Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis. J Biol Chem 284, 33740–33749
Herget, M., Kreissig, N., Kolbe, C., Schölz, C., Tampé, R., and Abele, R.
(Siehe online unter https://doi.org/10.1074/jbc.M109.047779) - (2009) Structural arrangement of the transmission interface in the antigen ABC transport complex TAP. Proc Natl Acad Sci USA 106, 5551–5556
Oancea, G., O'Mara, M. L., Bennett, W. F. D., Tieleman, D. P., Abele, R., and Tampé, R.
(Siehe online unter https://doi.org/10.1073/pnas.0811260106) - (2010) Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation. Proc Natl Acad Sci USA 107, 9135–9140
Baldauf, C., Schrodt, S., Herget, M., Koch, J., and Tampé, R.
(Siehe online unter https://doi.org/10.1073/pnas.1001308107) - (2010) Tuning the cellular trafficking of the lysosomal peptide transporter TAPL by its N-terminal domain. Traffic 11, 383–393
Demirel, Ö., Bangert, I., Tampé, R., and Abele, R.
(Siehe online unter https://doi.org/10.1111/j.1600-0854.2009.01021.x) - (2011) Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus. J Biol Chem 286, 7104–7115
Zutz, A., Hoffmann, J., Hellmich, U. A., Glaubitz, C., Ludwig, B., Brutschy, B., and Tampé, R.
(Siehe online unter https://doi.org/10.1074/jbc.m110.201178) - (2011) Conformation of peptides bound to the transporter associated with antigen processing (TAP). Proc Natl Acad Sci USA 108, 1349–1354
Herget, M., Baldauf, C., Schölz, C., Parcej, D., Wiesmüller, K.-H., Tampé, R., Abele, R., and Bordignon, E.
(Siehe online unter https://doi.org/10.1073/pnas.1012355108) - (2011) Specific lipids modulate the transporter associated with antigen processing (TAP). J Biol Chem 286, 13346–13356
Schölz, C., Parcej, D., Ejsing, C. S., Robenek, H., Urbatsch, I. L., and Tampé, R.
(Siehe online unter https://doi.org/10.1074/jbc.m110.216416) - (2014) An annular lipid belt is essential for allosteric coupling and viral inhibition of the antigen translocation complex TAP (transporter associated with antigen processing). J Biol Chem 289, 33098–33108
Eggensperger, S., Fisette, O., Parcej, D., Schäfer, L. V., and Tampé, R.
(Siehe online unter https://doi.org/10.1074/jbc.M114.592832) - (2014) Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter. Nat Commun 5, 5419
Grossmann, N., Vakkasoglu, A. S., Hulpke, S., Abele, R., Gaudet, R., and Tampé, R.
(Siehe online unter https://doi.org/10.1038/ncomms6419) - (2015) Single liposome analysis of peptide translocation by the ABC transporter TAPL. Proc Natl Acad Sci USA 112, 2046–2051
Zollmann, T., Moiset, G., Tumulka, F., Tampé, R., Poolman, B., and Abele, R.
(Siehe online unter https://doi.org/10.1073/pnas.1418100112) - (2015) Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter. Nature 517, 396–400
Kim, J., Wu, S., Tomasiak, T. M., Mergel, C., Winter, M. B., Stiller, S. B., Robles- Colmanares, Y., Stroud, R. M., Tampé, R., Craik, C. S., and Cheng, Y.
(Siehe online unter https://doi.org/10.1038/nature13872) - (2016) Antigenic peptide recognition on the human ABC transporter TAP resolved by DNP-enhanced solid-state NMR spectroscopy. J Am Chem Soc 138, 13967–13974
Lehnert, E., Mao, J., Mehdipour, A. R., Hummer, G., Abele, R., Glaubitz, C., and Tampé, R.
(Siehe online unter https://doi.org/10.1021/jacs.6b07426) - (2017) Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP. Proc Natl Acad Sci USA 114, E438-E447
Nöll, A., Thomas, C., Herbring, V., Zollmann, T., Barth, K., Mehdipour, A. R., Tomasiak, T. M., Brüchert, S., Joseph, B., Abele, R., Oliéric, V., Wang, M., Diederichs, K., Hummer, G., Stroud, R. M., Pos, K. M., and Tampé, R.
(Siehe online unter https://doi.org/10.1073/pnas.1620009114) - (2017) Structure and dynamics of antigenic peptides in complex with TAP. Front Immunol 8, 10
Lehnert, E., and Tampé, R.
(Siehe online unter https://doi.org/10.3389/fimmu.2017.00010) - (2018) Moving the cellular peptidome by transporters. Front Cell Dev Biol 6, 43
Abele, R., and Tampé, R.
(Siehe online unter https://doi.org/10.3389/fcell.2018.00043)
