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Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier

ABSTRACT: Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

SUBMITTER: Dr. Nanami Senoo

PROVIDER: S-SCDT-10_1038-S44318-024-00132-2 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

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Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier

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