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

Abstract

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.

Investigating the Broad Matrix-Gate Network in the Mitochondrial ADP/ATP Carrier through Molecular Dynamics Simulations

“…Considering consensus sequence feature at the triplet 79 positions in contrast to the tremendous diversity in structures and charges of the substrates transported by mitochondrial carriers, we infer that the triplet 79 positions could be more related to the general transport mechanism shared by this family, rather than carrying out substrate discrimination function. This inference is also supported by our recent identification of a new highly specific ADP binding site near the upper region of the cavity in c-state AAC [ 17 ].…”

“…However, K22 does not become involved in forming salt bridges with other residues in wild-type apo AAC [ 4 ], and therefore we did not investigate the impact of K22 in the current work. MD simulations on ADP binding process suggest that K22 might help catch the ADP detached from the specific binding site and relay it to the central binding site [ 17 ].…”

“…Our previous work showed that due to the dynamic property of H6, the [YWF][KR]G motif in domain 3 lost its β-turn structure in one of the three 3-μs trajectories on wild-type apo AAC, and Y228 before the kink proline P229 becomes the only barrier to separate the solvent in the pocket from the bulk solvent in the matrix side [ 17 ]. This observation also indicates the vulnerability of the interface between domains 3 and 1.…”

“…The multiple sequence alignment of SLC25A43 was calculated based on 171 sequences from the UniProt database. The results of other carriers were obtained from our recent work [ 17 ]. The residues are numbered based on hAAC1 (human ADP/ATP carrier), SLC25A42, SLC25A16, SCaMC1, SLC25A41 and SLC25A43, respectively.…”