Structural dynamics of the mitochondrial ADP/ATP carrier support an asymmetric transport mechanism

The mitochondrial ADP/ATP carrier (AAC) mediates ADP/ATP exchange across inner mitochondrial membrane via alternating between cytosol-open (c-) and matrix-open (m-) states. Despite the determination of crystallized structures for both states, its transport mechanism still remains unclear. One obstacle is that the structures are co-crystallized with bulky and asymmetric inhibitors. Here, we carried out molecular dynamics simulations on the m-state AAC with and without the co-crystallized nanobody and inhibitor BKA. We found that in apo AAC, the matrix side exhibits enhanced asymmetry, suggesting that, contrary to the previous proposal, the asymmetry observed in the crystallized structure is not an artifact of inhibitor binding. Comparative analysis of apo AAC trajectories in m- and c-states reveals that transmembrane helix H2 undergoes the most drastic conformational changes, while the H4-H5 bundle shows the least change among neighboring helix pairs. Further analysis indicates that the PG-level 1 in H2 and H6 separates the carrier into two asymmetric motion units, with the smaller unit undergoing more pronounced movement. Mutagenesis experiments validate functional significance of the asymmetric distribution of highly conserved Px[D/E]xx[K/R] and [D/E]G motifs among three repeat domains. Free energy calculations further confirm the previously reported asymmetric internal interactions in c-state AAC despite its pseudo-symmetric structure. Collectively, these results point to an asymmetric transport mechanism of AAC. Moreover, the work sheds light on the special internal three-repeat topology of the mitochondrial carrier family, and underscores the necessity of incorporating structural dynamics data when deducing transport mechanisms from static experimental structures in the two states.