The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix. The carrier cycles by an unresolved mechanism between the cytoplasmic state, in which the carrier accepts ADP from the cytoplasm, and the matrix state, in which it accepts ATP from the mitochondrial matrix. Here we present the structures of the yeast ADP/ATP carriers Aac2p and Aac3p in the cytoplasmic state. The carriers have three domains and are closed at the matrix side by three interdomain salt-bridge interactions, one of which is braced by a glutamine residue. Glutamine braces are conserved in mitochondrial carriers and contribute to an energy barrier, preventing the conversion to the matrix state unless substrate binding occurs. At the cytoplasmic side a second salt-bridge network forms during the transport cycle, as demonstrated by functional analysis of mutants with charge-reversed networks. Analyses of the domain structures and properties of the interdomain interfaces indicate that interconversion between states involves movement of the even-numbered α-helices across the surfaces of the odd-numbered α-helices by rotation of the domains. The odd-numbered α-helices have an L-shape, with proline or serine residues at the kinks, which functions as a lever-arm, coupling the substrate-induced disruption of the matrix network to the formation of the cytoplasmic network. The simultaneous movement of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins. These findings provide a structural description of transport by mitochondrial carrier proteins, consistent with an alternating-access mechanism.membrane protein | cardiolipin binding | X-ray crystallography | serine kinks | adenine nucleotide translocase M itochondrial carriers are a family of proteins that transport a diverse range of nucleotides, amino acids, inorganic ions, fatty acids, keto acids, and cofactors across the inner mitochondrial membrane (1). The carriers link the biochemical pathways in the cytoplasm with those in the mitochondrial matrix, thereby playing key roles in many aspects of cell physiology. There are many rare, but severe, human diseases associated with defective mitochondrial carriers (2).The ADP/ATP carriers are archetypal members of the mitochondrial carrier family (3). ADP/ATP carriers play the essential role of importing ADP into the mitochondrial matrix, where it can be phosphorylated by ATP synthase, and of exporting newly synthesized ATP into the cytosol, replenishing the cell with metabolic energy. ADP/ATP carriers have been intensively studied, because of their high natural abundance and the availability of specific inhibitors, which lock the carrier in two distinct states. The atractylosides, such as carboxyatractyloside (CATR) (4, 5), lock the carrier in the cytosolic state (c-state) with the substrate-binding site accessible to the intermembrane space, which is confluent with the cytosol. Bongkrekic acid (6) locks the carrier in the matrix state (m-state), wit...

Mitochondrial ADP/ATP carriers are inhibited by two natural compounds, atractyloside (ATR) or carboxy-atractyloside (CATR), which differ by one carboxylate group. The interactions of the inhibitors with the carrier were investigated by single-molecule force spectroscopy. Transmembrane alpha helices of the ATR-inhibited carrier displayed heterogeneous mechanical and kinetic properties. Whereas alpha helix H2 showed the most brittle mechanical properties and lowest kinetic stability, alpha helix H5 was mechanically the most flexible and possessed a kinetic stability 9 orders of magnitude greater than that of alpha helix H2. In contrast, CATR-binding substantially increased the kinetic stability of alpha helix H2 and tuned the mechanical flexibility of alpha helices H5 and H6. NMR spectroscopy confirmed that the additional carboxylate group of CATR binds to the sixth alpha helix, indicating that the enhanced stability of H2 is mediated via interactions between CATR and H6.

No abstract