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Ahmet Vakkasoglu (l) and Rachelle Gaudet

At the heart of our adaptive immune system is the presentation of internal antigens, usually peptides, to cytotoxic T cells. The Transporter associated with Antigen Processing (TAP) transports the small antigen peptides across the endoplasmic reticulum membrane where they are loaded onto major histocompatibility (MHC) class I molecules and travel to the cell surface for screening by T cells. Therefore TAP has a central role in fighting infections (particularly viral infections) and deregulated cells such as cancer cells. TAP uses the energy provided by the cellular fuel, ATP, to pump these antigen peptides across lipid membranes. But many aspects of how such nanoscale machinery functions are still unknown. In particular, how does the transporter pump peptides only in one direction and against a gradient – allowing the peptides to essentially swim upstream? In this study (Grossman N., Vakkasoglu A.S., Hulpke S., Abele R., Gaudet R., Tampe R., Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter, Nature Communications, 2014) in collaboration with Dr. Robert Tampé and his colleagues at Goethe University, Frankfurt, we make use of a mutant with an unusual behavior to better understand, at a molecular level, how TAP transports peptides unidirectionally.

We identified a mutation in a highly conserved sequence motif – called the D-loop – that decouples the two main activities of TAP: ATP hydrolysis and peptide transport. This mutation, within the ATP-hydrolyzing engine, changes a negatively-charged aspartate (or “D” of the D-loop) into a smaller and uncharged alanine. Although this mutant TAP was not able to hydrolyze ATP, it still transported peptides across the membrane, but in either direction, as long as it was down a gradient or “downstream”. Thus, while ATP hydrolysis is not necessary for peptide transport across the membrane, it is required for unidirectional transport. In other words, TAP spends ATP to insure that it transports antigen peptides in the correct direction and with high enough efficiency to yield optimal antigen screening by the immune system.

We further investigated the molecular details causing the mutant behavior. TAP functions as a heterodimer – it consists of two similar, but not identical, halves. The affinity between two subunits is important for its function. We crystallized and determined the structure of the mutant ATP-hydrolyzing engine domain, which turned out to be very similar to wild type version in overall structure, except that its D-loop region was more flexible or dynamic. Furthermore, our biophysical measurements showed that the affinity between the two subunits is dramatically decreased by the mutation. Therefore, although the TAP heterodimer can still form, it is looser. It also can come apart more readily; more specifically, it no longer requires ATP hydrolysis to come apart. This removes a checkpoint in the transport process, a checkpoint that normally insures that the peptides flow strictly from the inside to the outside of the cell.

In summary, our results lead us to propose a new model for the transport mechanism of molecular transport machinery like TAP where ATP hydrolysis serves as a checkpoint that enables unidirectional transport. There are nearly 50 similar transporters in the human genome, several of which are associated with diseases such as cancer, cystic fibrosis and retinal degeneration. Our results therefore have important implications for how we think about both the normal and disease-causing mechanisms of these important machines that transport a wide variety of molecules across cellular membranes.

Read more in Nature Communications or download PDF

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