In 2008, we proposed a new transport mechanism for mitochondrial carriers based on the disruption and formation of the two salt bridge networks on either side of the substrate binding site. This alternating access mechanism involves the simultaneous rotation of three domains around the central substrate translocation pathway, which is a unique mechanism for transport proteins. The structural mechanism of transport will explain the molecular basis of the diseases associated with dysfunctional mitochondrial carriers
Figure Mitochondrial carriers cycle between a matrix and cytoplasmic state by formation and disruption of the cytoplasmic salt bridge network (top) and matrix salt bridge network (bottom).
Mitochondrial carriers function as monomers , meaning that the previously proposed mechanisms based on the dimeric structure were no longer tenable . Mitochondrial carriers have a well conserved three-fold pseudo-symmetry in their sequences and structures , which must be important for the structural mechanism of transport . Highly conserved symmetrical residues were found to flank the central substrate binding site at the water-membrane interfaces. One set consists of the three PX[DE]XX[RK] signature motifs, which form a salt bridge network on the matrix side of the cavity, when the substrate binding site is open to the mitochondrial intermembrane space . The second set has three symmetrical [FY][DE]XX[RK] motifs on the cytoplasmic side of the cavity, which forms a salt bridge network when the substrate binding site is accessible from the mitochondrial matrix . The opening and closing of the carrier to the cytoplasmic and matrix side of the membrane is coupled to the disruption and formation of the two salt bridge networks. Recently, we have provided experimental evidence showing that the postively and negatively charged residues of the cytoplasmic network interact in the transport cycle  and that the cytoplasmic salt bridge network forms in the matrix state by using thermostability studies of carriers locked in the matrix or cytoplasmic state by inhibitors . The structures of the mitochondrial ADP/ATP carrier in the cytoplasmic state and the matrix state have shown that this structural mechanism indeed holds   .
We want to resolve the molecular basis of mitochondrial diseases associated with mitochondrial carriers by determining the structural mechanism of transport.
Figure The thermostability of the bongkrekic acid-inhibited (BKA) matrix state is dependent on the interaction energy of the cytoplasmic salt bridge network, whereas the carboxyatractyloside-inhibited (CATR) cytoplasmic state is not . ADP was added to obtain a homogeneous population trapped in a single state.
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