ADP/ATP Carrier Research Articles

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

The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.

AAC as a Potential Target Gene to Control Verticillium dahliae.

Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier) is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs) targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC). Compared with wild-type V. dahliae (Vd wt), ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C). Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

VDAC regulates AAC-mediated apoptosis and cytochrome c release in yeast.

Mitochondrial outer membrane permeabilization is a key event in apoptosis processes leading to the release of lethal factors. We have previously shown that absence of the ADP/ATP carrier (AAC) proteins (yeast orthologues of mammalian ANT proteins) increased the resistance of yeast cells to acetic acid, preventing MOMP and the release of cytochrome c from mitochondria during acetic acid - induced apoptosis. On the other hand, deletion of POR1 (yeast voltage-dependent anion channel - VDAC) increased the sensitivity of yeast cells to acetic acid. In the present work, we aimed to further characterize the role of yeast VDAC in acetic acid - induced apoptosis and assess if it functionally interacts with AAC proteins. We found that the sensitivity to acetic acid resulting from POR1 deletion is completely abrogated by the absence of AAC proteins, and propose that Por1p acts as a negative regulator of acetic acid - induced cell death by a mechanism dependent of AAC proteins, by acting on AAC - dependent cytochrome c release. Moreover, we show that Por1p has a role in mitochondrial fusion that, contrary to its role in apoptosis, is not affected by the absence of AAC, and demonstrate that mitochondrial network fragmentation is not sufficient to induce release of cytochrome c or sensitivity to acetic acid - induced apoptosis. This work enhances our understanding on cytochrome c release during cell death, which may be relevant in pathological scenarios where MOMP is compromised.

Specific Lipid Binding of Membrane Proteins in Detergent Micelles Characterized by NMR and Molecular Dynamics.

Many membrane proteins bind specifically to lipids as an integral component of their structures. The ability of detergents to support lipid binding is thus an important consideration when solubilizing membrane proteins for structural studies. In particular, the zwitterionic phosphocholine (PC)-based detergents, which have been widely used in solution NMR studies of channels and transporters, are controversial because of their strong solubilization power and thus perceived as more denaturing than nonionic detergents such as the maltosides. Here, we investigate the ability of the mitochondrial ADP/ATP carrier (AAC) to specifically bind cardiolipin, a mitochondrial lipid important for the carrier function, in dodecylphosphocholine (DPC) micelles. We found that in DPC, the AAC specifically binds cardiolipin in a manner consistent with the bound cardiolipins found in the crystal structures of the AAC determined in n-decyl β-d-maltoside. Our results suggest that PC detergent is compatible with specific lipid binding and that PC detergent mixed with the relevant lipid represents a viable solubilization system for NMR studies of membrane proteins.

The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes

The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978–2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.

Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion.

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters that have 2-fold direct or inverted quasi-symmetry, AAC has the apparent 3-fold rotational symmetry. Further, its transport rate is fast for transporters that carry large solutes. Here, we perform comprehensive NMR relaxation dispersion measurements for the yeast AAC carrier 3, which provide residue-specific information on the protein conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a lowly populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP significantly increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that while the transporter catalyzes the translocation of substrate, the substrate also facilitates interconversion between alternating states that may be relevant to the transport function.

Molecular Basis of MgATP Selectivity of the Mitochondrial SCaMC Carrier

The mitochondrial matrix is the supplier of cellular ATP. The short Ca(2+)-binding mitochondrial carrier (SCaMC) is one of the two mitochondrial carriers responsible for transporting ATP across the mitochondrial inner membrane. While the ADP/ATP carrier (AAC) accounts for the bulk ADP/ATP recycling in the matrix, the function of SCaMC is important for mitochondrial activities that depend on adenine nucleotides, such as gluconeogenesis and mitochondrial biogenesis. A key difference between SCaMC and AAC is that SCaMC selectively transports MgATP whereas AAC only transports free nucleotides. Here, we use a combination of nuclear magnetic resonance experiments and functional mutagenesis to investigate the structural basis of the MgATP selectivity in SCaMC. Our data revealed an MgATP binding site inside the transporter cavity, while identifying an aspartic acid residue that plays an important role in the higher selectivity for MgATP over free ATP.

A presequence-binding groove in Tom70 supports import of Mdl1 into mitochondria

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry gate into mitochondria for almost all imported proteins. A variety of specific receptors allow the TOM complex to recognize targeting signals of various precursor proteins that are transported along different import pathways. Aside from the well-characterized presequence receptors Tom20 and Tom22 a third TOM receptor, Tom70, binds proteins of the carrier family containing multiple transmembrane segments. Here we demonstrate that Tom70 directly binds to presequence peptides using a dedicated groove. A single point mutation in the cavity of this pocket (M551R) reduces the presequence binding affinity of Tom70 ten-fold and selectively impairs import of the presequence-containing precursor Mdl1 but not the ADP/ATP carrier (AAC). Hence Tom70 contributes to the presequence import pathway by recognition of the targeting signal of the Mdl1 precursor.

The ADP/ATP carrier and its relationship to oxidative phosphorylation in ancestral protist trypanosoma brucei.

The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.

β-Biguanidinium-cyclodextrin: a supramolecular mimic of mitochondrial ADP/ATP carrier protein

We reported a novel mono-β-cyclodextrin derivative, mono-6-deoxy-6-biguanidino-β-cyclodextrin (β-biGCD), which was investigated as a mimic of ADP/ATP carrier (AAC). Its affinity toward AMP, ADP, and ATP was evaluated by means of isothermal titration calorimetry (ITC). The association constants (Ka) of β-biGCD binding to AMP, ADP, and ATP were determined to be (1.07±0.04)×106, (5.86±0.02)×106, and (4.33±0.06)×106 L mol−1, respectively, which were 100-fold higher than mono-guanidino-β-cyclodextrin (ca. 104 L mol−1). UV spectroscopic titrations further confirmed the above results. The interaction between β-biGCD and nucleotides was probed by docking simulation. These results reveal that the biguanidinium moiety mimics the arginine residues of mitochondrial AAC protein.

Unveiling the last missing link of the cardiolipin synthetic pathway in mitochondria

Mitochondria produce cellular energy ATP by using electrochemical energy stored as a proton gradient across the mitochondrial inner membrane (IM). To generate the proton gradient, the respiratory-chain complexes in the IM pump up protons from the matrix to the inter-membrane space by coupling it to the electron transport through the respiratory chain. The efficient electron flow relies on the respiratory-chain super-complex formation, which is facilitated by the mitochondrial signature phospholipid cardiolipin (CL) [1]. CL directly interacts with the respiratory-chain complexes, mitochondrial carrier proteins including ADP/ATP carrier (AAC) etc. and stabilizes the super complexes consisting of Complexes I, III and IV in mammals and Complexes III and IV with AAC in yeast [1]. In addition, it is known that the assembly and maintenance of the TIM23 complex, a mitochondrial protein translocator in the IM, depends on the presence of CL [2]. Although the electron transport is essential for energy production in mitochondria, it could also generate reactive oxygen species (ROS), which are the potential cause of the oxidative damage of DNA and proteins as well as phospholipids in mitochondria. Accumulation of oxidative damage is thought to lead to mitochondrial and cellular dysfunction associated with aging [3]. CL is one of the main targets of ROS in mitochondria due to its IM location close to the sites of ROS production and its feature containing high-levels of unsaturated fatty acids [3]. Therefore CL could be a key molecule in aging process. CL is synthesized through several steps of modifications of phosphatidic acid (PA) in the IM and is remodeled by deacylation and re-acylation that generate mature CL with unsaturated fatty acids [1]. Despite its physiological importance, it was not clear how CL is synthesized in mitochondria. In particular, an enzyme responsible for the first step of the CL synthesis, i.e. generation of an important intermediate phospholipid, CDP-diacylglycerol (CDP-DAG), in mitochondria was not identified. In the issue of Cell Metabolism (Volume 17, Issue 5, 709-718, 7 May 2013), we reported identification of Tam41 as a mitochondrial CDP-DAG synthase and the cellular system to compensate the loss of Tam41. Tam41 was originally identified as a protein important for assembly and maintenance of the TIM23 complex in the IM [4]. However, a subsequent study showed that loss of Tam41 leads to significant decrease in CL and accumulation of PA, the precursor phospholipid of CDP-DAG [5]. Besides, respiratory-chain super complexes tend to be destabilized in cells lacking Tam41, which indicates that the role of Tam41 is not limited to the TIM23 complex but to the protein complexes in the IM in general [5]. We thus reasoned that Tam41 could be a mitochondrial CDP-DAG synthase and asked whether Tam41 directly catalyzes the CDP-DAG formation. To this end, we purified Tam41 and its loss-of-function mutants from yeast cells and measured their CDP-DAG synthase activities in vitro. We observed generation of CDP-DAG upon incubation of wild-type Tam41, but not the Tam41 mutants, with fluorescence-labeled PA and CTP. These results demonstrate that Tam41 is a mitochondrial CDP-DAG synthase, which had been elusive previously (Figure ​(Figure11). Figure 1 Roles of Tam41 and Art5 in yeast hospholipid biosynthetic pathways Our previous study identified Art5 as a multi-copy suppressor of tam41Δ cells [6]. Art5 is a member of the arrestin-related trafficking adaptor (ART) protein family and is known to facilitate degradation of inositol transporter in plasma membrane in response to exogenous inositol [7]. Indeed, we found that growth defects of tam41Δ cells were restored in inositol-depleted media (Figure ​(Figure1).1). Surprisingly, despite the restoration of the growth defects of tam41Δ cells by overexpressing Art5, the low level of CL hardly changed while the CDP-DAG, PA, and PE (phophatidylethanolamine) levels increased and the PI (phosphatidylinositol) level decreased. Hence CL itself is not critical for cell growth, but overall alteration of the phospholipid composition is capable of compensation of the loss of Tam41. This observation also points to the fact that yeast cells have a backup system to tolerate the loss of Tam41. In relation to this, Connerth et al. showed that defects in cell growths and decrease in the CL level caused by the loss of Tam41 can be largely restored by the simultaneous loss of a PA transfer protein Ups1 [8]. This suggests the presence of an alternative CL synthetic pathway, which may be activated in the absence of both Tam41 and Ups1. These findings may offer hints about possible treatments of diseases and protection against aging processes associated with mitochondrial dysfunction arising from the compromised CL synthesis or CL oxidation.

The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

Impaired Transport of Nucleotides in a Mitochondrial Carrier Explains Severe Human Genetic Diseases

The mitochondrial ADP/ATP carrier (AAC) is a prominent actor in the energetic regulation of the cell, importing ADP into the mitochondria and exporting ATP toward the cytoplasm. Severe genetic diseases have been ascribed to specific mutations in this membrane protein. How minute, well-localized modifications of the transporter impact the function of the mitochondria remains, however, largely unclear. Here, for the first time, the relationship between all documented pathological mutations of the AAC and its transport properties is established. Activity measurements combined synergistically with molecular-dynamics simulations demonstrate how all documented pathological mutations alter the binding affinity and the translocation kinetics of the nucleotides. Throwing a bridge between the pathologies and their molecular origins, these results reveal two distinct mechanisms responsible for AAC-related genetic disorders, wherein the mutations either modulate the association of the nucleotides to the carrier by modifying its electrostatic signature or reduce its conformational plasticity.

Rcf1 and Rcf2, Members of the Hypoxia-Induced Gene 1 Protein Family, Are Critical Components of the Mitochondrial Cytochrome bc1-Cytochrome c Oxidase Supercomplex

We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.

Transport Cycle of Mitochondrial Carriers from Internal Symmetries

Mitochondrial carriers (MC) are nuclear-encoded transporters which exchange charged molecules across the inner membrane of mitochondria. The common transport cycle of structurally and functionally similar MCs involves transitions between two major cytoplasmic-open (c-) and matrix-open (m-) states. The only available structure of an MC is that of ADP/ATP carrier (AAC) in c-state. Previous computational and experimental work have characterized the binding site and functional conformational changes of AAC, but fallen short of explaining the full transport cycle. MCs are consisted of three structurally similar repeats each composed of two helices. Based on sequence alignments of individual helices across all MCs, a model for conformational changes resulting in m-state is suggested. The model implies that the helices kink or straighten around certain key residues involving prolines and glycines, so that helices in each repeat exchange conformations among each other. In order to get a detailed realistic view of the transport cycle and to obtain an atomic resolution m-state model of AAC, we combined homology modeling with non-equilibrium driven MD simulations. Initially, we generated crude models of the m-state AAC by modeling each helix in each repeat based on its partner helix. We used the crude m-state models as targets in targeted molecular dynamics (TMD) simulations. Besides, we applied time dependent harmonic restraints on certain collective variables reflecting the conformational change between the c-state and the model m-state. The collective variables include dihedrals of the key prolines and glycines along with rotational orientations of the pieces of helices with respect to the binding site region. Here, we present plausible m-state structures of AAC and transition pathways along with several intermediates identified from the common features of the non-equilibrium simulations requiring the lowest work.

Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier

We have previously shown that acetic acid activates a mitochondria-dependent death process in Saccharomyces cerevisiae and that the ADP/ATP carrier (AAC) is required for mitochondrial outer membrane permeabilization and cytochrome c release. Mitochondrial fragmentation and degradation have also been shown in response to this death stimulus. Herein, we show that autophagy is not active in cells undergoing acetic acid-induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we found that the vacuolar protease Pep4p and the AAC proteins have a role in mitochondrial degradation using yeast genetic approaches. Depletion and overexpression of Pep4p, an orthologue of human cathepsin D, delays and enhances mitochondrial degradation respectively. Moreover, Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment. AAC-deleted cells also show a decrease in mitochondrial degradation in response to acetic acid and are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast programmed cell death.

ミトコンドリア内膜のADP/ATP透過担体のC末端領域の構造特性と機能

Mitochondrial ADP/ATP carrier (AAC) is a protein catalyzing the transport of adenine nucleotides across inner mitochondrial membrane. In this review article, we first briefly introduce structural and functional properties of this protein. Next, we describe the results of our recent studies on the difference in the C-terminal region between yeast type 2 AAC isoform and bovine type 1 AAC isoform. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier are discussed.

High-Chloride Concentrations Abolish the Binding of Adenine Nucleotides in the Mitochondrial ADP/ATP Carrier Family

The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.

A first low-resolution difference Fourier map of phosphorus in a membrane protein from near-edge anomalous diffraction

Crystal diffraction of three membrane proteins (cytochrome bc(1) complex, sarcoplasmic reticulum Ca(2+) ATPase, ADP-ATP carrier) and of one nucleoprotein complex (leucyl tRNA synthetase bound to tRNAleu, leuRS:tRNAleu) was tested at wavelengths near the X-ray K-absorption edge of phosphorus using a new set-up for soft X-ray diffraction at the beamline ID01 of the ESRF. The best result was obtained from crystals of Ca(2+) ATPase [adenosin-5'-(beta,gamma-methylene) triphosphate complex] which diffracted out to 7 A resolution. Data were recorded at a wavelength at which the real resonant scattering factor of phosphorus reaches the extreme value of -20 electron units. The positions of the four triphosphates of the monoclinic unit cell of the ATPase have been obtained from a difference Fourier synthesis based on a limited set of anomalous diffraction data.

Molecular Basis of Substrate Selectivity in the ADP/ATP Carrier

The ADP/ATP carrier (AAC) is a membrane transporter that mediates the exchange of ADP and ATP across the mitochondrial inner membrane. During an exchange cycle, AAC switches between two conformational states, the cytoplasm-open state (c-state), and the matrix-open state (m-state). Our recent molecular dynamics simulations revealed spontaneous binding of ADP to the c-state AAC, and identified the unknown binding site for ADP as a pocket deeply positioned inside the lumen that forms through significant conformational changes of several basic residues in response to substrate binding. We also showed that ADP binding likely triggers AAC transition to the m-state by breaking a salt bridge network. The identified binding site has allowed us to explore substrate selectivity in AAC by simulating the “binding” of various ligands, e.g., AMP and Mg-ADP, to AAC. AMP does bind but is not transported by AAC, and Mg is known to have an inhibitory effect on AAC. However, the molecular details involved in these processes are largely unknown. Our results suggest that the presence of a minimum of two phosphate groups in their Mg-free form is absolutely necessary for proper binding and for initiating the structural changes required for activation of AAC. AMP and Mg-ADP cannot establish sufficient contact with the salt bridge ring at the bottom of AAC lumen, either due to lack of the beta-phosphate in AMP, or interference of Mg2+ with the phosphate groups in Mg-ADP. These results provide additional evidence for the ADP binding site characterized in our earlier study, and suggest a mechanism for substrate selectivity in AAC.