The substrate specificity of the human ADP/ATP carrier AAC1

Abstract

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

The transport mechanism of the mitochondrial ADP/ATP carrier

“…Because of the crystal symmetry, the crystal in a dioleoyl phosphocholine bilayer contains rows of Aac3p crystallographic dimers with matrix side up or down orientations [74]. The substrate binding site of AAC1 identified by (A) chemical and distance constraints [90,91], by (B) symmetry analysis [92], and by (C) and (D) molecular dynamics simulations [87,89,93]. The key residues are shown as red sticks, except for (B), where the sticks are coloured according to their symmetry score from highly asymmetric (red) to neutral (white) to highly symmetric (blue).…”

“…Recently, the substrate specificity of the human carrier was determined [89]. Remarkably, only ADP and ATP and their deoxy variants are transported.…”

“…The third approach used molecular dynamics simulations to identify residues of the bovine mitochondrial ADP/ATP carrier that are involved in the trajectory and binding of ADP in the cavity [87,89,93]. Although ADP binds in many places along the trajectory, two consensus sites emerged from these three studies.…”

Dangerous Liaisons between Detergents and Membrane Proteins. The Case of Mitochondrial Uncoupling Protein 2

“…It is widely used to investigate the behavior of membrane proteins in a lipid environment and, hence, can complement structural data obtained in detergent solution. MD was recently employed to study the mitochondrial AAC, confirming that the structure solved in the presence of a specific inhibitor is also capable of binding spontaneously its endogenous nucleotide substrates 19,32,33 . Here, we show that UCP2 behaves as a pore in DPC, but rapidly collapses in a lipid environment, suggesting that the structure of UCP2 in DPC does not reflect the membrane-bound, functional conformation.…”

Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations

“…The IF structure obtained is characterized by newly formed well-packed and -organized inter-domain interactions and was shown to be stable during MD simulations for several microseconds. The simulation also shed light on a mechanism of the strict bidirectional transport selectivity of adenosine nucleotides over guanosine and inosine ones [23–25]. The IF form was then shown to bind ATP and BA and thus revealed their binding mechanisms.…”