Exploring the multifaceted role of adenosine nucleotide translocase 2 in cellular and disease processes: A comprehensive review.

Adenosine nucleotide translocases (ANTs) are a family of proteins abundant in the inner mitochondrial membrane, primarily responsible for shuttling ADP and ATP across the mitochondrial membrane. Additionally, ANTs are key players in balancing mitochondrial energy metabolism and regulating cell death. ANT2 isoform, highly expressed in undifferentiated and proliferating cells, is implicated in the development and drug resistance of various tumors. We conduct a detailed analysis of the potential mechanisms by which ANT2 may influence tumorigenesis and drug resistance. Notably, the significance of ANT2 extends beyond oncology, with roles in non-tumor cell processes including blood cell development, gastrointestinal motility, airway hydration, nonalcoholic fatty liver disease, obesity, chronic kidney disease, and myocardial development, making it a promising therapeutic target for multiple pathologies. To better understand the molecular mechanisms of ANT2, this review summarizes the structural properties, expression patterns, and basic functions of the ANT2 protein. In particular, we review and analyze the controversy surrounding ANT2, focusing on its role in transporting ADP/ATP across the inner mitochondrial membrane, its involvement in the composition of the mitochondrial permeability transition pore, and its participation in apoptosis.

The essential role of adenine nucleotide translocase 4 on male reproductive function in mice.

Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.

FA Sliding as the Mechanism for the ANT1-Mediated Fatty Acid Anion Transport in Lipid Bilayers.

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.

The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.

Human mitochondrial ADP/ATP carrier SLC25A4 operates with a ping-pong kinetic mechanism.

The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.

The ant's weapon improves honey bee learning performance.

Formic acid is the main component of the ant's major weapon against enemies. Being mainly used as a chemical defense, the acid is also exploited for recruitment and trail marking. The repelling effect of the organic acid is used by some mammals and birds which rub themselves in the acid to eliminate ectoparasites. Beekeepers across the world rely on this effect to control the parasitic mite Varroa destructor. Varroa mites are considered the most destructive pest of honey bees worldwide and can lead to the loss of entire colonies. Formic acid is highly effective against Varroa mites but can also kill the honeybee queen and worker brood. Whether formic acid can also affect the behavior of honey bees is unknown. We here study the effect of formic acid on sucrose responsiveness and cognition of honey bees treated at different live stages in field-relevant doses. Both behaviors are essential for survival of the honey bee colony. Rather unexpectedly, formic acid clearly improved the learning performance of the bees in appetitive olfactory conditioning, while not affecting sucrose responsiveness. This exciting side effect of formic acid certainly deserves further detailed investigations.

Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

Mitochondrial protein import clogging as a mechanism of disease.

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.

Inside our cells, compartments known as mitochondria generate the chemical energy required for life processes to unfold. Most of the proteins found within mitochondria are manufactured in another part of the cell (known as the cytosol) and then imported with the help of specialist machinery. For example, the TOM and TIM22 channels provide a route for the proteins to cross the two membrane barriers that separate the cytosol from the inside of a mitochondrion. ANT1 is a protein that is found inside mitochondria in humans, where it acts as a transport system for the cell's energy currency. Specific mutations in the gene encoding ANT1 have been linked to degenerative conditions that affect the muscles and the brain. However, it remains unclear how these mutations cause disease. To address this question, Coyne et al. recreated some of the mutations in the gene encoding the yeast equivalent of ANT1 (known as Aac2). Experiments in yeast cells carrying these mutations showed that the Aac2 protein accumulated in the TOM and TIM22 channels, creating a 'clog' that prevented other essential proteins from reaching the mitochondria. As a result, the yeast cells died. Mutant forms of the human ANT1 protein also clogged up the TOM and TIM22 channels of human cells in a similar way. Further experiments focused on mice genetically engineered to produce a "super-clogger" version of the mouse equivalent of ANT1. The animals soon developed muscle and neurological conditions similar to those observed in human diseases associated with ANT1. The findings of Coyne et al. suggest that certain genetic mutations in the gene encoding the ANT1 protein cause disease by blocking the transport of other proteins to the mitochondria, rather than by directly affecting ANT1's nucleotide trnsport role in the cell. This redefines our understanding of diseases associated with mitochondrial proteins, potentially altering how treatments for these conditions are designed.

Lipopolysaccharide administration increases the susceptibility of mitochondrial permeability transition pore opening via altering adenine nucleotide translocase conformation in the mouse liver.

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces various biological reactions in vivo. Our previous study suggested that LPS administration disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in the liver mitochondria, and sensitizes mitochondrial permeability transition (MPT) pore opening in rats. However, it is unknown whether LPS-induced MPT pore opening in rats is similarly observed in mice and whether the mechanism is the same. LPS administration to mice increased not only cyclosporin A-sensitive swelling (MPT pore opening) susceptibility, but also induced cyclosporin A-insensitive basal swelling, unlike in rats. In addition, respiratory activity observed after adding ADP was significantly decreased. Based on these results, we further investigated the role of adenine nucleotide translocase (ANT). Carboxyatractyloside (CATR; an ANT inhibitor) treatment decreased respiratory activity after ADP was added in vehicle-treated mitochondria similarly to LPS administration. Additionally, CATR treatment increased MPT pore opening susceptibility in LPS-treated mitochondria compared to that of vehicle-treated mitochondria. Our study shows that ANT maintained a c-state conformation upon LPS administration, which increased MPT pore opening susceptibility in mice. These results suggest that LPS enhances MPT pore opening susceptibility across species, but the mechanism may differ between rat and mouse.

KH-17, a simplified derivative of bongkrekic acid, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane.

Two natural products, bongkrekic acid and carboxyatractyloside, are known to specifically inhibit the mitochondrial ADP/ATP carrier from its matrix side and cytosolic side, respectively, in concentration ranges of 10-6  M. In the present study, we investigated the manner of action of a synthetic bongkrekic acid derivative, KH-17, lacking three methyl groups, one methoxy group, and five internal double bonds, on the mitochondrial ADP/ATP carrier. At slightly acidic pH, KH-17 inhibited mitochondrial [3 H]ADP uptake, but its inhibitory action was about 10 times weaker than that of its parental compound, bongkrekic acid. The main site of action of KH-17 was confirmed as the matrix side of the ADP/ATP carrier by experiments using submitochondrial particles, which have an inside-out orientation of the inner mitochondrial membrane. However, when we added KH-17 to mitochondria at neutral pH, it had a weak inhibitory effect on [3 H]ADP uptake, and its inhibitory strength was similar to that of bongkrekic acid. These results indicated that KH-17 weakly inhibits the ADP/ATP carrier not only from the matrix side but also from the cytosolic side. To ascertain whether this interpretation was correct, we examined the effects of KH-17 and carboxyatractyloside on mitochondrial [3 H]ADP uptake at two [3 H]ADP concentrations. We found that both KH-17 and carboxyatractyloside showed a stronger inhibitory effect at the lower [3 H]ADP concentration. Therefore, we concluded that the bongkrekic acid derivative, KH-17, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane. These results suggested that the elimination of three methyl groups, one methoxy group, and five internal double bonds present in bongkrekic acid altered its manner of action towards the mitochondrial ADP/ATP carrier. Our data will help to improve our understanding of the interaction between bongkrekic acid and the mitochondrial ADP/ATP carrier.

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families.

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

Function-Related Asymmetry of the Interactions between Matrix Loops and Conserved Sequence Motifs in the Mitochondrial ADP/ATP Carrier.

The ADP/ATP carrier (AAC) plays a central role in oxidative metabolism by exchanging ATP and ADP across the inner mitochondrial membrane. Previous experiments have shown the involvement of the matrix loops of AAC in its function, yet potential mechanisms remain largely elusive. One obstacle is the limited information on the structural dynamics of the matrix loops. In the current work, unbiased all-atom molecular dynamics (MD) simulations were carried out on c-state wild-type AAC and mutants. Our results reveal that: (1) two ends of a matrix loop are tethered through interactions between the residue of triplet 38 (Q38, D143 and Q240) located at the C-end of the odd-numbered helix and residues of the [YF]xG motif located before the N-end of the short matrix helix in the same domain; (2) the initial progression direction of a matrix loop is determined by interactions between the negatively charged residue of the [DE]G motif located at the C-end of the short matrix helix and the capping arginine (R30, R139 and R236) in the previous domain; (3) the two chemically similar residues D and E in the highly conserved [DE]G motif are actually quite different; (4) the N-end of the M3 loop is clamped by the [DE]G motif and the capping arginine of domain 2 from the two sides, which strengthens interactions between domain 2 and domain 3; and (5) a highly asymmetric stable core exists within domains 2 and 3 at the m-gate level. Moreover, our results help explain almost all extremely conserved residues within the matrix loops of the ADP/ATP carriers from a structural point of view. Taken together, the current work highlights asymmetry in the three matrix loops and implies a close relationship between asymmetry and ADP/ATP transport.

Identification of ADP/ATP Translocase 1 as a Novel Glycoprotein and Its Association with Parkinson's Disease.

Protein glycosylation plays a crucial role in central nervous system, and abnormal glycosylation has major implications for human diseases. This study aims to evaluate an etiological implication of the variation in glycosylation for Parkinson's disease (PD), a neurodegenerative disorder. Based on a PD mouse model constructed by the intraperitoneal injection with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, glycosylation variation was accessed using biotinylated lectin of dolichos biflorus agglutinin (DBA) specific for the exposed N-acetylgalactosamine linked to glycoprotein. Consequently, a glycoprotein with a significantly reduced N-acetylgalactosamination was identified as ADP/ATP translocase 1 (ANT1) by lectin affinity chromatography coupled with MALDI-TOF MS/MS (mass spectrometry), and confirmed by the analysis of dual co-immunofluorescence and Western blot. A tissue-specific distribution of de-N-acetylgalactosaminated ANT1 was found to be correlated with high risk of PD. At cellular level, an obvious co-aggregation between ANT1 and DBA was only found in the MPP+-induced PD-like cell model using dual co-immunofluorescence. Thus, we found that ANT1 was a potential glycoprotein with terminal N-acetylgalactosamine moiety, and the variation of glycosylation in ANT1 was associated with PD. This investigation provides an innovative insight in protein glycosylation with PD pathogenesis.

Alkyl esters of 7-hydroxycoumarin-3-carboxylic acid as potent tissue-specific uncouplers of oxidative phosphorylation: Involvement of ATP/ADP translocase in mitochondrial uncoupling.

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation - alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

Reversible modification of mitochondrial ADP/ATP translocases by paired Legionella effector proteins.

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria's benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.

Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle.

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state.

Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs. Our results show that on the current microsecond simulation time scale: 1) CL binding does not significantly affect overall stability of the carrier or structural symmetry at the matrix-gate level; 2) pocket volumes of the carrier and interactions involved in the matrix-gate network become more heterogeneous in parallel simulations with membranes containing CLs; 3) CL binding consistently strengthens backbone hydrogen bonds within helix H2 near the matrix side; and 4) CLs play a consistent stabilizing role on the domain 1-2 interface through binding with the R30:R71:R151 stacking structure and fixing the M2 loop in a defined conformation. CL is necessary for the formation of this stacking structure, and this structure in turn forms a very stable CL binding site. Such a delicate equilibrium suggests the strictly conserved R30:R71:R151stacking structure of AACs could function as a switch under regulation of CLs. Taken together, these results shed new light on the CL-mediated modulation of AAC function.

Novel US-CpHMD Protocol to Study the Protonation-Dependent Mechanism of the ATP/ADP Carrier.

We have designed a protocol combining constant-pH molecular dynamics (CpHMD) simulations with an umbrella sampling (US) scheme (US-CpHMD) to study the mechanism of ADP/ATP transport (import and export) by their inner mitochondrial membrane carrier protein [ADP/ATP carrier (AAC)]. The US scheme helped overcome the limitations of sampling the slow kinetics involved in these substrates' transport, while CpHMD simulations provided an unprecedented realism by correctly capturing the associated protonation changes. The import of anionic substrates along the mitochondrial membrane has a strong energetic disadvantage due to a smaller substrate concentration and an unfavorable membrane potential. These limitations may have created an evolutionary pressure on AAC to develop specific features benefiting the import of ADP. In our work, the potential of mean force profiles showed a clear selectivity in the import of ADP compared to ATP, while in the export, no selectivity was observed. We also observed that AAC sequestered both substrates at longer distances in the import compared to the export process. Furthermore, only in the import process do we observe transient protonation of both substrates when going through the AAC cavity, which is an important advantage to counteract the unfavorable mitochondrial membrane potential. Finally, we observed a substrate-induced disruption of the matrix salt-bridge network, which can promote the conformational transition (from the C- to M-state) required to complete the import process. This work unraveled several important structural features where the complex electrostatic interactions were pivotal to interpreting the protein function and illustrated the potential of applying the US-CpHMD protocol to other transport processes involving membrane proteins.