Structural Basis of Substrate Recognition by the Mitochondrial ADP/ATP Transporter

Specific import of ADP and export of ATP by ADP/ATP carrier (AAC) across the inner mitochondrial membrane are crucial for sustainable energy supply in all eukaryotes. However, mechanism for highly specific substrate recognition in the dynamic transport process remains largely elusive. Here, unguided MD simulations of 22 microseconds in total reveal that AAC in ground c-state uses the second basic patch (K91K95R187), tyrosine ladder (Y186Y190Y194), F191 and N115 in the upper region of the cavity to specifically recognize ADP and confer selectivity for ADP over ATP. Mutations of these residues in yeast AAC2 reduce ADP transport across the L. lactis membrane and induce defects in OXPHOS and ATP production in yeast. Sequence analyses also suggest that AAC and other adenine nucleotide transporters use the upper region of the cavity, rather than the central binding site to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC, and together with central binding site support early biochemical findings about existence of two substrate binding sites. Our results imply that using different sites for substrate recognition and conformational transition could be a smart strategy for transporters to cope with substrate recognition problem in the highly dynamic transport process.