Human mitochondrial ADP/ATP carrier SLC25A4 operates with a ping-pong kinetic mechanism
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ABSTRACT:
SUBMITTER: Ms. Camila Cimadamore-Werthein
PROVIDER: S-SCDT-10_15252-EMBR_202357127 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.
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Project description:Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.
| S-SCDT-10_1038-S44318-024-00150-0 | biostudies-other
Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.
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Project description:Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane alpha-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-methanethiosulfonate (MTSES), whereas the activity of a mutant AAC2, devoid of cysteines, is unaffected. Wild-type and cysteine-less AAC2 were coexpressed in different molar ratios in yeast mitochondrial membranes. After addition of MTSES the residual transport activity correlated linearly with the fraction of cysteine-less carrier present in the membranes, and so the two versions functioned independently of each other. Also, the cysteine-less and wild-type carriers were purified separately, mixed in defined ratios and reconstituted into liposomes. Again, the residual transport activity in the presence of MTSES depended linearly on the amount of cysteine-less carrier. Thus, the entire transport cycle for ADP/ATP exchange is carried out by the monomer.
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Project description:For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.
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Project description:The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.
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Project description:Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of dodecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins is preserved in DPC, thereby suggesting that DPC is a suitable environment in which to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of cardiolipins to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investigation in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment.
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Project description:The mitochondrial ADP/ATP carrier, also called adenine nucleotide translocase, accomplishes one of the most important transport activities in eukaryotic cells, importing ADP into the mitochondrial matrix for ATP synthesis, and exporting ATP to fuel cellular activities. In the transport cycle, the carrier changes between a cytoplasmic and matrix state, in which the central substrate binding site is alternately accessible to these compartments. A structure of a cytoplasmic state was known, but recently, a structure of a matrix-state in complex with bongkrekic acid was solved. Comparison of the two states explains the function of highly conserved sequence features and reveals that the transport mechanism is unique, involving the coordinated movement of six dynamic elements around a central translocation pathway.
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Project description:DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.
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Project description:piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5' Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.
| S-EPMC3463270 | biostudies-literature