Mitochondrial carriers transport organic acids, amino acids, nucleotides and cofactors across the mitochondrial inner membrane. These transporters consist of a three-fold symmetric bundle of six transmembrane α-helices that encircle a pore with a central substrate binding site, whose alternating access is controlled by a cytoplasmic and a matrix gate (C- and M-gates). The C- and M-gates close by forming two different salt-bridge networks involving the conserved motifs [YF][DE]XX[KR] on the even-numbered and PX[DE]XX[KR] on the odd-numbered transmembrane α-helices, respectively. We have investigated the effects on transport of mutating the C-gate charged residues of the yeast NAD⁺ transporter Ndt1p and performed molecular docking with NAD⁺ and other substrates into structural models of Ndt1p. Double-cysteine substitutions and swapping the positions of the C-gate charged-pair residues showed that all of them contribute to the high transport rate of wild-type Ndt1p, although no single salt bridge is essential for activity. The in silico docking results strongly suggest that both the C-gate motif mutations and our previously reported M-gate mutations affect gate closing, whereas those of the M-gate also affect substrate binding, which is further supported by molecular dynamics. In particular, NAD⁺ most likely interferes with the cation-π interaction between R303-W198, which has been proposed to exist in the Ndt1p M-gate in the place of one of the salt bridges. These findings contribute to understanding the roles of the charged C- and M-gate residues in the transport mechanism of Ndt1p.

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Journal Article

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Miniero DV, Palmieri F, Quadrotta V, Polticelli F, Palmieri L, Monné M

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