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Principal Investigator

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P16  Robert Tampé
Professor & Director

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Institute of Biochemistry
Biocenter
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29475
Fax +49 (0)69 798-29495

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P16 Translocation mechanism of ABC exporters and the peptide-loading complex

 

The transporter associated with antigen processing (TAP) is a focal point in adaptive immunity against infected or malignantly transformed cells. The heterodimeric ABC transporter shuttles proteasomal degradation products into the endoplasmic reticulum (ER) for loading onto major histocompatibility complex class-I (MHC-I) molecules. A macromolecular peptide loading complex (PLC), consisting of TAP1/2, the chaperones tapasin, calreticulin, ERp57, MHC-I heavy chain, and β2-microglobulin, orchestrates the recognition, translocation, editing, and final transfer of peptides to MHC-I. As major breakthroughs of the current funding period, we delineated the mechanistic basis of the directionality and energetics of the TAP complex and we specified the stoichiometry, interaction principles, as well as the mechanistic mode of action of key components of the PLC. Furthermore, we unveiled two hitherto unknown inhibition mechanisms of how two unrelated viral factors encoded by herpesvirus and poxvirus inhibit TAP by different strategies and thereby escape immune surveillance. Finally, we determined the first structure of an ABC transporter by single-particle cryo-EM in a unique asymmetric inward-open conformation.

In the upcoming funding period, we aim at investigating the newly identified trans-inhibition mechanism, conformational states, and dynamics of the TAP and related ABC transporters. We will study the architecture of the translocation machineries at different levels of complexity using viral inhibitors, antibody fragments, and strategic mutations to arrest the ABC transporters in defined states for structural and mechanistic studies. The proposal focuses on the translocation and trans-inhibition mechanisms of the TAP complex and related ABC transporters, the identification of different conformational states and their allosteric coupling during the translocation cycle. We will address the structural organization of the PLC and its inhibition by viral immune evasion proteins using an integrative structural and functional approach. The PLC is probably the most complex machinery in antigen processing known and, with millions of substrates, the scope of its multitasking functions is likewise unparalleled.

 

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Publications

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Kim JM, Wu, S, Tomasiak T, Mergel C, Winter MN, Stiller S, Robles-Colmanares Y, Stroud RM*, Tampé R*, Craik CS*, Cheng Y* (2015) Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter. Nature 517, 396-400.

Bechara C, Nöll A, Morgner, N, Degiacomi, MT, Tampé R*, Robinson CV* (2015) A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nature Chemistry 7, 255-62. 

Lehnert E, Mao J, Mehdipour AR, Hummer S, Abele R, Glaubitz C, Tampé R (2016) Antigenic peptide recognition on the human ABC transporter TAP resolved by DNP-enhance solid-state NMR spectroscopy. J Am Chem Soc, in press.

Blees A, Reichel K, Trowitzsch S, Fisette O, Bock C, Abele R, Hummer G, Schäfer LV, Tampé R (2015) Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch. Sci Rep 5, 17341. 

Fleischmann G, Fisette O, Thomas C, Wieneke R, Tumulka F, Schneeweiss C, Springer S, Schäfer LV, Tampé R (2015) Mechanistic basis for epitope proofreading in the peptide-loading complex. J Immunol 195, 4503-13.
– Highlighted by Cutting Edge Article: J Immunol 195, 4041-2.

Eggensperger S, Tampé R (2015) The transporter associated with antigen processing –
a key player in adaptive immunity. Biol Chem 396, 1059-72. 

Trowitzsch S, Tampé R (2015) Multicolor fluorescence-based screening toward structural analysis of multiprotein membrane complexes. Methods Enzymol 557, 3-26. 

Zollmann T, Moiset G, Tumulka F, Tampé R, Poolman B, Abele R (2015) Single liposome analysis of peptide translocation by the ABC transporter TAPL. Proc Natl Acad Sci USA 112, 2046-51. 

Fischbach H, Döring M, Nikles D, Lehnert E, Baldauf C, Kalinke U, Tampé R (2015) Ultra-sensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets. Nature Commun 6, 6199.

Lin J, Eggensperger S, Hank S, Wieneke R, Mayerhofer PU, Tampé R (2014) A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome. PLoS Pathogens 10, e1004554. 

Grossmann N, Vakkasoglu AS, Hulpke S, Abele R, Gaudet R, Tampé R (2014) Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter. Nature Commun 5, 5419.

Hinz, A., Jedamzick, J., Herbring, V., Fischbach, H., Hartmann, J., Parcej, D., Koch, J. and Tampé, R. (2014) Assembly and function of the MHC I peptide-loading complex are conserved across jawed vertebrates. J Biol Chem 289, 33109-33117. 


Eggensperger S, Fisette O, Parcej D, Schäfer VL, Tampé R (2014) An annular lipid belt is essential for allosteric coupling and viral inhibition of the antigen translocation complex TAP. J Biol Chem 289, 33098-108.

Seyffer F, Tampé R (2014) ABC transporters in adaptive immunity. Biochim Biophys Acta 1850, 449-460.

Mayerhofer PU, Tampé R (2014) Antigen translocation machineries in adaptive immunity and viral immune evasion. J Mol Biol 427, 1102-18. 

Nair-Gupta P, Baccarini A, Tung N, Seyffer F, Florey O, Huang, Y, Banerjee M, Overholtzer M, Roche PA, Tampé R, Brown DB, Amsen D, Whiteheart SW, Blander JM (2014) TLR signals induce phagosomal MHC-I delivery from endosomal recycling compartment to allow cross-presentation. Cell 185, 506-514.

Hulpke S, Tampé R (2013) The MHC I loading complex: a multitasking machinery in adaptive immunity. Trends Biochem Sci 38, 412-420. 

Parcej D, Guntrum R, Schmidt S, Hinz A, Tampé R (2013) Multicolour fluorescence-detection size-exclusion chromatography for structural genomics of membrane multiprotein complexes. PLOS One 8, e67112. 

Hulpke S, Baldauf C, Tampé R (2012) Molecular architecture of the MHC I peptide-loading complex - one tapasin molecule is essential and sufficient for antigen processing. FASEB J 26, 5071-80.

*corresponding authors

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Collaborations

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Kühlbrandt (P01), Glaubitz (P06), Prisner/Joseph (P07), Abele (P09), Heilemann (P20), Geertsma (P23), Morgner (P24), Hummer (P25)