Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between