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Electrostatic funneling of ADP binding. ( a ) ADP binding process revealed by simulation NB4. ADP molecules at t ϭ 0, 0.7, 3.2, and 100.0 ns are colored from light to dark red, respectively. The 1.0-V electrostatic potential isosurface calculated from the apo-AAC simulation is shown in a blue mesh. R235 is shown in vdW representation. ( b ) Superposition of NB1, NB2, and NB4 at t ϭ 5 ns. Despite the different initial orientations, ADP molecules in the three simulations adopt similar final orientations, and all form salt bridges with K22, R79, and R279. ( c ) Orientation of ADP in the first 10-ns simulations
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Exchange of ATP and ADP across mitochondrial membrane replenishes the cytoplasm with newly synthesized ATP and provides the mitochondria with the substrate ADP for oxidative phosphorylation. The sole means of this exchange is the mitochondrial ADP/ATP carrier (AAC), a membrane protein that is suggested to cycle between two conformationally distinct...
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... found to diffuse spontaneously toward the bottom of the AAC lumen over a distance of 20 Å, penetrating deeply into the basin within only a few nanoseconds. Despite opposite initial orienta- tions, all AAC-bound ADP molecules converge into similar final orientations, in which phosphate groups are pointing down and inserted into the binding site (Fig. 2). The only exception is simulation NB3, in which ADP was attracted by two basic residues (R187 and K95) near the entrance of the vestibule and, thus, did not succeed in reaching the bottom of the basin in 15 ns of simulation (Fig. S1). We have also performed a 10-ns simulation of ATP binding (ATP-AAC, Table S1) in which an ATP molecule ...
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... a time scale comparable with ADP, consistent with the fact that both ADP and ATP can bind the c-state AAC and the direction of transport is merely determined by the electrochemical gradient of the nucleo- tides (4). ADP binding appears to be steered primarily by the phosphate groups. This process captured in detail for simulation NB4 is shown in Fig. 2a (also see animations in Movie S1 and Movie S2). At t 5 ns, ADP molecules in three of the four simulations (NB1, NB2, and NB4) have approximately reached the bottom of the vestibule and established similar interaction patterns with the protein. The phosphate groups form multiple salt bridges with residues R79, R279, and K22 (Fig. 2b). ...
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... NB4 is shown in Fig. 2a (also see animations in Movie S1 and Movie S2). At t 5 ns, ADP molecules in three of the four simulations (NB1, NB2, and NB4) have approximately reached the bottom of the vestibule and established similar interaction patterns with the protein. The phosphate groups form multiple salt bridges with residues R79, R279, and K22 (Fig. 2b). The orientation of ADP, defined as the angle between the membrane normal and a vector pointing from the -phosphorus atom to the center of mass of ADP, stabilizes 30 o at t 6 ns in all three simulations (Fig. 2c). Note that no external forces or biasing potentials are used during these simulations. Therefore, the observed, extremely ...
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... similar interaction patterns with the protein. The phosphate groups form multiple salt bridges with residues R79, R279, and K22 (Fig. 2b). The orientation of ADP, defined as the angle between the membrane normal and a vector pointing from the -phosphorus atom to the center of mass of ADP, stabilizes 30 o at t 6 ns in all three simulations (Fig. 2c). Note that no external forces or biasing potentials are used during these simulations. Therefore, the observed, extremely rapid binding of ADP in multiple simulations reflects an active participation of the carrier in recruiting the substrate. Below, we show that unique electrostatic features of AAC are the main driving force for the ...
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... such a potential at the surface of the protein. In the absence of ADP (apo-AAC), Cl ions, which are initially placed in the bulk solvent to neutralize the simulation system, bind to the carrier within a few nanoseconds; at t 3 ns, one Cl ion is found to enter the AAC vestibule, whereas two others adhered to the surface of AAC from the matrix side (Fig. S2). During the 55-ns-simulation, up to three Cl ions are found in the lumen simultaneously, the average number of luminal Cl being 1.9. However, these Cl ions do not show any specific binding and dynamically interact with multiple basic residues in the lumen. The two surface Cl ions, on the other hand, visit specific sites when they ...
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... basic residues in the lumen. The two surface Cl ions, on the other hand, visit specific sites when they adhere to the surface of the protein. Interestingly, these sites are occupied by cardiolipin (negative) head groups in the crystal structure (8). The calculated electro- static potential identifies these sites as strongly positive regions (Fig. S2c). This is in close agreement with the results of NMR experiments indicating that a large number of cardiolipins stay tightly bound to AAC even after isolation of the protein from the membrane (22,23). Therefore, another role of the strong electrostatic potential in AAC might be in its insertion into the inner mitochondrial membrane that ...
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We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet signifi...
The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we...
Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, th...
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Citations
... The importance of the central arginine ring for the binding of ATP, ADP, and fatty acid anion has already been demonstrated for ANT, UCP1, and UCP3 [24,41,44,62,63]. We have hypothesized that the substrate binds in the same region of mUCP3. ...
Uncoupling protein 3 (UCP3) belongs to the mitochondrial carrier protein superfamily SLC25 and is abundant in brown adipose tissue (BAT), the heart, and muscles. The expression of UCP3 in tissues mainly dependent on fatty acid oxidation suggests its involvement in cellular metabolism and has drawn attention to its possible transport function beyond the transport of protons in the presence of fatty acids. Based on the high homology between UCP2 and UCP3, we hypothesized that UCP3 transports C4 metabolites similar to UCP2. To test this, we measured the transport of substrates against phosphate (32Pi) in proteoliposomes reconstituted with recombinant murine UCP3 (mUCP3). We found that mUCP3 mainly transports aspartate and sulfate but also malate, malonate, oxaloacetate, and succinate. The transport rates calculated from the exchange of 32Pi against extraliposomal aspartate and sulfate were 23.9 ± 5.8 and 17.5 ± 5.1 µmol/min/mg, respectively. Using site-directed mutagenesis, we revealed that mutation of R84 resulted in impaired aspartate/phosphate exchange, demonstrating its critical role in substrate transport. The difference in substrate preference between mUCP2 and mUCP3 may be explained by their different tissue expression patterns and biological functions in these tissues.
... To assess whether the ANT1 mutations affect the protein-mediated substrate binding, we measured the time course of the 3 H-ATP concentration in liposomes in the absence of the protein and in the presence of reconstituted ANT1 or mutant ( Figure S4). The relative ratios (k Mutant /k ANT1 ) show that mutations had only a slight effect on the ANT1mediated ADP/ATP exchange rate, k, (Figure 1e, Table S1), apart from ANT1-R59S, which is involved in attracting AA − but neither in binding the substrates nor in the matrix salt bridge network [16,24]. ...
... AA − Slides along a Positively Charged Surface at the Protein-Lipid Interface ANT1 has a considerable positive surface potential at the protein-lipid interface (Figure 1b). The positive potential extends deep into the center of the bilayer membrane and includes the substrate-binding center in the protein interior [16]. We had previously constructed trajectory density maps using a series of short unbiased MD simulations in which the AA − was randomly placed in the vicinity of the ANT1 surface along the positively charged electrostatic potential of ANT1 (see Figure 1b from [18]). ...
... Next, we identified a second FA − binding site-R79-in the substrate binding site of ANT1 ( Figure 6) [60,61]. This is of particular interest because the substrates (ADP and ATP) and the inhibitors (CATR and BKA) use R79 as a common binding target [16,17,26,[62][63][64]. Not only does ADP/ATP exchange negatively regulate FA transport but FA − and the substrates (CATR and BKA) also compete for R79 ( Figure 5). ...
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.
... However, a general mechanism of transport by mitochondrial carriers has been proposed based on atomic structures of the related ADP/ATP carrier (AAC) 5 . In the C-state conformation of AAC induced by carboxyatractyloside (CATR) 6,7 , the cavity is widely open toward the cytoplasmic side, allowing ADP to enter the positively charged electrostatic funnel 8,9 . Within the common substrate binding site located in the middle of the cavity, ADP is stabilized by electrostatic interactions with positively charged amino acids. ...
... At the end of the cavity, one helix turn away from triplet FIW88, the phosphate moiety of the nucleotide forms a stable interaction with triplet RRR84, which corresponds to triplet RGR88 of AAC also documented as a nucleotide anchor point 33,34 . Similar interactions have been observed in simulations of ADP binding to AAC 8,9 and UCP2 22 . ...
... All three arginines are known to be essential for UCP1 inhibition 29,37,38 : indeed, we find that the phosphate moiety of GDP or GTP consistently binds all three arginine residues (Fig. 4). In contrast, in AAC, asymmetric placement of the two arginines determines an asymmetric position of the nucleotide 8,9,34 . In a recent conceptual model of nucleotide translocation by AAC, the small size of G199 leaves a gap that can accommodate the base moiety (Figure 2a in ref. 5). ...
Brown adipose tissue expresses uncoupling protein 1 (UCP1), which dissipates energy as heat, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Our molecular simulations predict that GDP and GTP bind UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with conserved residues R92 and E191. We identify a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. In yeast spheroplast respiration assays, both I187A and W281A mutants increase the fatty acid-induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by fatty acids even at high concentrations of purine nucleotides. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.
... Given the potential biomedical interest, the current work aims to characterize the dynamics of the SLC25A20 through an in silico approach. In this regard, MD simulations have been frequently used to investigate the molecular mechanism of the MCF members [15][16][17][18][19][20][21][22][23]. In this work, AlphaFold2 has been used to predict a structural model of the cstate of the transporter. ...
... Indeed, several studies in recent years suggested that residues located at different depths of the carrier cavity establish interactions with the substrate, helping its migration deeper into the cavity [2,[24][25][26]. An early ligand recognition mechanism has also been hypothesized for the ADP/ATP carrier on the basis of MD simulations [17,22], confirmed by experimental studies [27]. ...
The Carnitine-Acylcarnitine Carrier is a member of the mitochondrial Solute Carrier Family 25 (SLC25), known as SLC25A20, involved in the electroneutral exchange of acylcarnitine and carnitine across the inner mitochondrial membrane. It acts as a master regulator of fatty acids β-oxidation and is known to be involved in neonatal pathologies and cancer. The transport mechanism, also known as “alternating access”, involves a conformational transition in which the binding site is accessible from one side of the membrane or the other. In this study, through a combination of state-of-the-art modelling techniques, molecular dynamics, and molecular docking, the structural dynamics of SLC25A20 and the early substrates recognition step have been analyzed. The results obtained demonstrated a significant asymmetry in the conformational changes leading to the transition from the c- to the m-state, confirming previous observations on other homologous transporters. Moreover, analysis of the MD simulations’ trajectories of the apo-protein in the two conformational states allowed for a better understanding of the role of SLC25A20 Asp231His and Ala281Val pathogenic mutations, which are at the basis of Carnitine-Acylcarnitine Translocase Deficiency. Finally, molecular docking coupled to molecular dynamics simulations lend support to the multi-step substrates recognition and translocation mechanism already hypothesized for the ADP/ATP carrier.
... This implies that without this positive charge, even with ideal positioning of the ring, the transport process may be halted. This model is consistent with the electrostatic funneling observed with ADP/ATP carriers (39)(40)(41)(42)(43)(44). Moreover, previous studies have found that AMP, dUTP, NADH, GDP and GTP could bind to the ADP/ATP carrier, suggesting that the dynamics of transport in cells involves an additional differentiation step to discriminate between bound molecules (45, 46). ...
SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that have been attributed to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD ⁺ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we used Molecular Dynamic simulations to study reconstituted SLC25A51 homology models in lipid bilayers. We observed spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51’s central pore and found that mutation of these sites impaired transporter activity. We also observed that stable formation of the required matrix gate was controlled by a single salt bridge. Using simulation data and in-cell activity assays we identified binding sites in SLC25A51 for NAD ⁺ and showed that its binding was guided by an electrostatic interaction between NAD ⁺ and a negatively charged patch in the pore. In turn, interaction of NAD ⁺ with interior residue E132 guided the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.
Significance
NAD ⁺ is an intermediary metabolite whose multiple functions are entwined with respiration, catabolism, and stress responses in cells. Previous sensor measurements had indicated that its continuous biosynthesis was required to sustain mitochondrial matrix levels in respiring cells, and SLC25A51 was identified as the required importer of NAD ⁺ across the inner mitochondrial membrane. However, SLC25A51 has little homology to other nucleotide carriers at its substrate binding site. By combining modeling approaches and experimental assays, this work provides mechanistic insight into how human SLC25A51 recognizes its ligand, how the transporter can be regulated by its lipid environment, and an observation of ligand-induced gate opening. This represents the first description of the ligand binding site for an NAD ⁺ mitochondrial carrier.
... ADP is an important molecule which is converted to adenosine diphosphate (ATP), the energy form used by cells for many enzymatic reactions and for the transportation of hormones, neurotransmitters and other molecules. (Wang & Tajkhorshid, 2008). Moreover, the antifolate resistance pathway describes how cells may develop resistance to antifolates, which act as antagonists of folic acid. ...
Phenotypic changes in response to environmental cues allow organisms to adapt and enhance their fitness in a given habitat. Despite the significance of phenotypic plasticity in the evolution and ecology of natural populations and the ongoing development of new genomic tools, the underlying genetic basis is still largely unknown. Herein, we examined the underlying mechanisms of genetic and phenotypic divergence among alternative morphs of a natural population of the Greek smooth newt (Lissotriton graecus). The studied population consists of fully aquatic individuals exhibiting facultative paedomorphosis, the retention of larval traits such as gills, and individuals that have passed metamorphosis (paedomorphic vs. metamorphic newts). Based on the single nucleotide polymorphisms (SNPs) obtained, we observed low genetic divergence between the two alternative morphs and similar levels of gene diversity on neutral markers. Despite the observed high gene flow between the morphs, an Fst approach for outliers detected candidate loci putatively associated with the alternative morphs that mapped to four genes. These identified genes have functional roles in metabolic processes that may mediate the persistence of alternative ontogenetic trajectories.
... FA cycling model also describes the activation and inhibition of FA-mediated H + transport by ANT1 (Fig. 1a) 6 , but the molecular details of the mechanism by which ANT1 transports FAback to the intermembrane space have remained obscure. We have now used the crystallographic structure of ANT1 16,17 and a series of MD simulations 18,19 to design specific recombinant ANT1 mutants and analyze the transport of FA -. The work uncovers the molecular mechanism by which ANT1 transports FAs. ...
... Fig. 1 and Suppl. Table 1), which is involved neither in binding the substrates nor in the matrix salt bridge network 18,20 . Measurements of membrane current revealed that the total conductance of membranes reconstituted with the K48S, R59S, and K62S mutants in the presence of arachidonic acid (AA), GMutant, decreased by 70 % but was almost not affected in K51 mutant ( Fig. 1f and Suppl. ...
... Previously we have shown that ANT1 has a considerable positive surface potential at the protein-lipid interface that is involved in binding AAon the matrix side 6 . It extends deep into the centre of the bilayer membrane and includes the substrate-binding centre of the protein 18 . To reveal the path for the AAtranslocation, we constructed density maps using a series of unbiased MD simulations in which the AAwas randomly placed in the vicinity of ANT1 along the positively charged electrostatic potential. ...
The additional protonophoric function of the mitochondrial adenine nucleotide translocase (ANT1) is now recognized. However, the molecular mechanism remains controversial. Fatty acid (FA) cycling hypothesis postulates that FAs transport protons across the inner mitochondrial membrane to the matrix by a flip-flop, whereas ANT1 facilitates the translocation of FA anions (FA ⁻ ) back to the intermembrane space. By a combined approach involving measurements of current through the planar lipid bilayers reconstituted with recombinant ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that FA ⁻ is initially caught by R59 on the matrix side of ANT1, then moves along the positively charged protein-lipid interface, and binds to R79, where it is protonated in the hydrated cavity in the presence of D134. R79 is crucial for the competitive binding of ANT1 substrates (ATP and ADP) and inhibitors (carboxyatractyloside, bongkrekic acid). The binding sites are well-conserved in mitochondrial SLC25 members, implying a general transporting mechanism for FA anions.
... Later, sequence analyses, which used chemical and distance constraints 18,19 or deviation of pseudosymmetry 20 as concepts, pointed towards a location in the central part of the cavity, which included three 'contact points' 18,19 ( Supplementary Fig. 1). In contrast, molecular dynamics simulations highlighted substrate interactions throughout the translocation pathway [28][29][30][31] . Evidently, the different studies have not pointed to a consensus location for the substrate-binding site, and thus its experimental determination will be an essential first step in addressing the key issues of substrate-binding and conformational coupling of the mitochondrial ADP/ATP carrier. ...
... Many proposals have been made for the location of the substrate-binding site 19,20,[24][25][26][27][28][29][30][31] , but here we address this question experimentally with the relevant substrates. Our approach allows a complete assessment of the substrate-binding process, highlighting only significant interactions. ...
... The observation that a single mutation can abolish binding altogether indicates that there are no other significant substratebinding sites elsewhere, such as the matrix loops or C-terminal region, as claimed previously [24][25][26][27] . It is possible that other observed interactions [28][29][30][31] are transient, including those of the "tyrosine ladder" 15,28 , for which we find no experimental support. Furthermore, since the thermostability shifts induced by ADP and ATP were not statistically different from each other at any concentration for any of the tested proteins ( Supplementary Fig. 7), both substrates must bind to the same set of residues in a similar way (Fig. 6). ...
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 trans-location. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.
... Both these salt bridge types are found in different positions in the three MCs studied and therefore, once again, these conclusions reflect a specific relationship between the particular MC and its substrate(s). In addition, our proposal that, in at least some MCs, the charged residues of the PX[DE]XX[RK] motifs are involved in interactions with the substrates is substantiated by available data from molecular dynamics and docking studies of Ctp1p, SLC25A4, Aac2p, and SLC25A29 [47,[70][71][72]. These data provide evidence that the substrates interact with the charged residues of the PX[DE]XX[RK] motifs and, in particular, with K37 and K239 (Ctp1p), K32 and D231 (SLC25A4), K33 and R236 (Aac2p), and D23, K26, E114, and D211 (SLC25A29). ...
Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.
... Principal component analysis also shows that major movements of AAC are observed in the region around the C2 loop and the cytoplasmic sides of H1 and H6, while the region around the C1 loop including cytoplasmic ends of H2 and H3 are relatively stable in both conditions with and without CLs (Fig. 2C, D). Our recent work had identified that the second basic patch (K91K95R187), N115 and the tyrosine ladder (Y186Y190Y194) including F191 form the specific ADP binding site (65), and ADP binding to this region was also reported by other groups (16,66,67). Thermodynamic stability of this region below the C1 loop could be crucial for efficient substrate recognition. ...
... In the crystal structures of CATR-bound AAC, charged residues from the Px[DE]xx[KR] motifs form a symmetrical and cyclic interhelical salt bridge network on the matrix side (m-gate) (70,71), and this network is proposed to be important to stabilize the c-state conformation (28). MD simulations from different groups consistently show that this network rearranges when the inhibitor CATR is removed (29,64,66,67,72). Based on simulations without CLs, we reported that after removing CATR, the charged residues from three Px[DE]xx[KR] motifs mainly form intrahelical salt bridges, and together with polar residues and positive residues K79, R235, and R279, form a broad asymmetric m-gate network (29). ...
... This result is consistent with our previous finding that R279 has little impact on other interactions of the m-gate network (73). The stable E29:R279 salt bridge was also reported in previous simulations on c-state AAC (66,72) and homologous SLC25A29 (D23:R257) (37), and the dissociation of E29:R279 is accompanied by the opening the matrix side during c-to m-state transition (74). ...
Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane (IMM). As the most abundant protein of the IMM, 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 (MD) 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.
