Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter: USE OF A TROJAN HORSE STRATEGY

Meike Herget,Giani Oancea,Susanne Schrodt,Michael Karas,Robert Tampé,Rupert Abele

doi:10.1074/jbc.m608480200

Meike Herget, Giani Oancea + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m608480200

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Abstract

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.

Highlights

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ATP-binding cassette (ABC) transporter transporter associated with antigen processing (TAP) plays a focal role in the adaptive immunity against infected or malignantly transformed cells
The transporter associated with antigen processing (TAP)4 plays a prominent role in the antigen-processing pathway via major histocompatibility complex (MHC) class I molecules [1, 2]
Peptides Modified by a Chemical Protease Bind to TAP—To identify residues within the TAP complex sensing incoming peptides, we developed a Trojan horse approach, where each position of the human leukocyte antigen-B27-restricted epitope RRYQKSTEL was modified by a small chemical protease via a single cysteine residue (C1- to C9-peptide, Fig. 1A)

Summary

Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter

We identified residues that are essential for tight coupling of peptide binding and transport This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotidebinding domains. By MALDITOF MS and cysteine cross-linking, the contact site was mapped to the cytosolic core loop 1 (CCL1) of TAP1 Within this loop, key residues were identified that upon exchange uncouple peptide binding from peptide transport. Key residues were identified that upon exchange uncouple peptide binding from peptide transport This transmission interface is structurally reorganized during the ATPhydrolysis cycle, demonstrating a critical function of this site in the inter-domain cross-talk within the TAP complex.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION