Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle
Description
Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes as part of oxidative phosphorylation. They cycle between two extreme states, the cytoplasmic- and matrix-open state, via a complex mechanism involving six structural elements, initiated by substrate binding. There was no experimental evidence to show which residues are involved in substrate binding, a key step of the mechanism. All 36 water-accessible residues in the substrate translocation pathway were mutated to alanine. Then, we applied two experimental approaches to identify their role in substrate binding. In the first one, we used complementation growth assays to identify residues that are important for the mechanism of the carrier, as substrate binding is a key aspect of it. For this purpose, a dilution series of yeast strains expressing the 36 single alanine replacement variants, as well as the wild type and empty vector control, were made on selective media containing glycerol. The spots were analysed by densitometry to assess the level of growth quantitatively and was used as a measure of functionality. The growth plates with the dilution series and the quantification data are submitted. In the second approach, we used thermostability shift assays to identify residues that are directly involved in substrate binding. It was noted previously that only the substrates ADP and ATP cause a shift in thermostability when they are bound to the carriers, using a CPM probe. This provided an opportunity to identify the residues involved, as mutations should lead to a reduction or abolishment of the shift. The substrate concentration-dependent shifts were measured for the wild type and all variants. The raw and derivative traces are submitted. We observe three classes: (i) variants of five positively charged residues, where the substrate concentration-dependent shift is completely abolished, indicating that they are critical for binding, (ii) variants of six polar, aliphatic and aromatic residues, where the size of the shift is significantly reduced, indicating that they make contributions to binding, and (iii) variants that have a similar shift as the wild type, indicating that they are not involved in binding. The residues that play critical and contributing roles in substrate binding all cluster together halfway the translocation pathway. Seven of them are accessible and have similar conformers in both states, forming the main binding site. The other four form two pairs of asparagine/arginine residues, positioned above and below the main binding site. These pairs are pointing towards the opening of the cavity in one state but are part of the main binding site in the other state, alternately. The results clearly show that substrate binding is a step-wise process, guiding the conformational changes of the carrier in an interdependent way. The properties of the site explain reversibility and electrogenicity of transport.