Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Abstract

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acidbAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

The Transmembrane Prolines of the Mitochondrial ADP/ATP Carrier Are Involved in Nucleotide Binding and Transport and Its Biogenesis

“…Pro-247 is located in the C-terminal part of TMH5, which displays the Ancp signature sequence, RRRMMM. This region of BfAnc1p exhibited similar accessibility to the solvent whatever the inhibitor bound (14), suggesting TMH5 is less flexible than its counterparts TMH1 and -3. This is in accordance with our results and with molecular dynamics experiments during which, movements around the prolyl residue in TMH5 are less important than movements around the prolyl residue in TMH1 (13).…”

“…This reorganization unlocks the three odd-numbered TMH allowing the protein to expand in the matrix half. Besides, deuterium exchange experiments carried out with the BfAnc1p showed that TMH1, -2, -3, and -5 were much less exposed to the solvent on the IMS side in the BA complex than in the CATR complex, indicating rearrangement of the two halves of the carrier: the one oriented through the matrix and the other one to the IMS, one relative to the other (14). Similar experiments carried out with yeast ScAnc2p evidenced inhibitor-dependent deuterium accessibility: the matrix loops were more deuterated in the presence of BA than in the presence of CATR (40).…”

“…They observed on the nanosecond time scale radial, outward displacements of the C-terminal part of the odd numbered helices around Pro-27, Pro-132, and Pro-229 (in TMH1, -3, and -5, respectively), accompanied by rearrangements of the salt bridge network responsible for the closure of the bottom of the cavity. Besides, hydrogen/deuterium exchange kinetics coupled to mass spectrometry highlighted dramatic accessibility to the solvent of the regions exposed to the matrix and to the intermembrane space depending on the inhibitor bound to BfAnc1p, CATR, or BA (14), in line with conformational dynamics during the transport. For example, regions 21-35 in TMH1 and 129 -140 in TMH3, encompassing MCF motives 1 and 2, were much more deuterated in the BA-carrier complex than in the CATR-carrier complex.…”

Yeast ADP/ATP Carrier Isoform 2

“…Bacteria were transformed according to standard methods using CaCl 2 (11 (14), and JL1-3ANC2⌬por1 (W303-1B anc1::LEU2 anc3:: URA3 por1::HIS3). 6 Strains were cultivated and transformed as described (15). A full description of the compositions of all media used, YPL (lactate-containing rich medium) and YNB Glc W Ϫ and YNB Gal W Ϫ (tryptophan-free minimal medium containing glucose or galactose, respectively) can be found in Clémençon et al (15).…”

“…Recently, both complexes were compared in detergent solution by hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS). These analyses provided new information on the BA conformation and, thus, on the conformational dynamics of bAnc1p during ADP/ATP transport (6). However, the bovine model remains limited to biochemical studies of the native form because its overexpression in bacteria or in yeast is difficult (7).…”