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.

Department of Mathematics Informatics and Cybernetics University of Chemistry and Technology 166 28 Prague Czech Republic

Division of Organic Chemistry and Biochemistry Rudjer Bošković Institute 10000 Zagreb Croatia

Institute of Physiology Pathophysiology and Biophysics Department of Biomedical Sciences University of Veterinary Medicine 1210 Vienna Austria

Zobrazit více v PubMed

Nicholls D.G. The bioenergetics of brown adipose tissue mitochondria. FEBS Lett. 1976;61:103–110. doi: 10.1016/0014-5793(76)81014-9. PubMed DOI

Krauss S., Zhang C.Y., Lowell B.B. The mitochondrial uncoupling-protein homologues. Nat. Rev. Mol. Cell Biol. 2005;6:248–261. doi: 10.1038/nrm1592. PubMed DOI

Zackova M., Skobisova E., Urbankova E., Jezek P. Activating omega-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3. J. Biol. Chem. 2003;278:20761–20769. doi: 10.1074/jbc.M212850200. PubMed DOI

Andreyev A., Bondareva T.O., Dedukhova V.I., Mokhova E.N., Skulachev V.P., Tsofina L.M., Volkov N.I., Vygodina T.V. The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria. Eur. J. Biochem. 1989;182:585–592. doi: 10.1111/j.1432-1033.1989.tb14867.x. PubMed DOI

Brustovetsky N., Klingenberg M. The Reconstituted Adp/Atp Carrier Can Mediate H+ Transport by Free Fatty-Acids, Which Is Further Stimulated by Mersalyl. J. Biol. Chem. 1994;269:27329–27336. doi: 10.1016/S0021-9258(18)46989-X. PubMed DOI

Kreiter J., Rupprecht A., Skulj S., Brkljaca Z., Zuna K., Knyazev D.G., Bardakji S., Vazdar M., Pohl E.E. ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport. Int. J. Mol. Sci. 2021;22:2490. doi: 10.3390/ijms22052490. PubMed DOI PMC

Bertholet A.M., Chouchani E.T., Kazak L., Angelin A., Fedorenko A., Long J.Z., Vidoni S., Garrity R., Cho J., Terada N., et al. H(+) transport is an integral function of the mitochondrial ADP/ATP carrier. Nature. 2019;571:515–520. doi: 10.1038/s41586-019-1400-3. PubMed DOI PMC

Urbankova E., Voltchenko A., Pohl P., Jezek P., Pohl E.E. Transport kinetics of uncoupling proteins. Analysis of UCP1 reconstituted in planar lipid bilayers. J. Biol. Chem. 2003;278:32497–32500. doi: 10.1074/jbc.M303721200. PubMed DOI

Beck V., Jaburek M., Demina T., Rupprecht A., Porter R.K., Jezek P., Pohl E.E. Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers. FASEB J. 2007;21:1137–1144. doi: 10.1096/fj.06-7489com. PubMed DOI

Macher G., Koehler M., Rupprecht A., Kreiter J., Hinterdorfer P., Pohl E.E. Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate. Biochim. Biophys. Acta Biomembr. 2018;1860:664–672. doi: 10.1016/j.bbamem.2017.12.001. PubMed DOI PMC

Pohl E.E., Rupprecht A., Macher G., Hilse K.E. Important Trends in UCP3 Investigation. Front. Physiol. 2019;10:470. doi: 10.3389/fphys.2019.00470. PubMed DOI PMC

Skulachev V.P. Fatty-Acid Circuit as a Physiological Mechanism of Uncoupling of Oxidative-Phosphorylation. FEBS Lett. 1991;294:158–162. doi: 10.1016/0014-5793(91)80658-P. PubMed DOI

Kamp F., Hamilton J.A. pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids. Proc. Natl. Acad. Sci. USA. 1992;89:11367–11370. doi: 10.1073/pnas.89.23.11367. PubMed DOI PMC

Kamp F., Zakim D., Zhang F., Noy N., Hamilton J.A. Fatty acid flip-flop in phospholipid bilayers is extremely fast. Biochemistry. 1995;34:11928–11937. doi: 10.1021/bi00037a034. PubMed DOI

Winkler E., Klingenberg M. Effect of fatty acids on H+ transport activity of the reconstituted uncoupling protein. J. Biol. Chem. 1994;269:2508–2515. doi: 10.1016/S0021-9258(17)41974-0. PubMed DOI

Wang Y., Tajkhorshid E. Electrostatic funneling of substrate in mitochondrial inner membrane carriers. Proc. Natl. Acad. Sci. USA. 2008;105:9598–9603. doi: 10.1073/pnas.0801786105. PubMed DOI PMC

Pebay-Peyroula E., Dahout-Gonzalez C., Kahn R., Trezeguet V., Lauquin G.J., Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature. 2003;426:39–44. doi: 10.1038/nature02056. PubMed DOI

Kreiter J., Brkljača Z., Škulj S., Bardakji S., Vazdar M., Pohl E.E. Mechanism of the ANT-mediated transport of fatty acid anions across the inner mitochondrial membrane. bioRxiv. 2022 doi: 10.1101/2022.06.27.497434. bioRxiv:2022.06.27.497434. DOI

Beck V., Jaburek M., Breen E.P., Porter R.K., Jezek P., Pohl E.E. A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1. Biochim. Biophys. Acta. 2006;1757:474–479. doi: 10.1016/j.bbabio.2006.03.006. PubMed DOI

Kreiter J., Beitz E., Pohl E.E. A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes. Biomolecules. 2020;10:685. doi: 10.3390/biom10050685. PubMed DOI PMC

Heidkamper D., Muller V., Nelson D.R., Klingenberg M. Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes. Biochemistry. 1996;35:16144–16152. doi: 10.1021/bi960668j. PubMed DOI

King M.S., Kerr M., Crichton P.G., Springett R., Kunji E.R.S. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier. Biochim. Biophys. Acta. 2016;1857:14–22. doi: 10.1016/j.bbabio.2015.09.013. PubMed DOI PMC

Kreiter J., Skulj S., Brkljaca Z., Zuna K., Vazdar M., Pohl E.E. The transport of fatty acid anions across the inner mitochondrial membrane by the adenine nucleotide translocase. Eur. Biophys. J. 2021;50((Suppl. S1)):S57. doi: 10.1016/j.bpj.2021.11.1180. DOI

Škulj S., Brkljača Z., Vazdar M. Molecular Dynamics Simulations of the Elusive Matrix-Open State of Mitochondrial ADP/ATP Carrier. Isr. J. Chem. 2020;60:735–743. doi: 10.1002/ijch.202000011. DOI

Bertholet A.M., Natale A.M., Bisignano P., Suzuki J., Fedorenko A., Hamilton J., Brustovetsky T., Kazak L., Garrity R., Chouchani E.T., et al. Mitochondrial uncouplers induce proton leak by activating AAC and UCP1. Nature. 2022;606:180–187. doi: 10.1038/s41586-022-04747-5. PubMed DOI PMC

Mifsud J., Ravaud S., Krammer E.M., Chipot C., Kunji E.R., Pebay-Peyroula E., Dehez F. The substrate specificity of the human ADP/ATP carrier AAC1. Mol. Membr. Biol. 2013;30:160–168. doi: 10.3109/09687688.2012.745175. PubMed DOI

Ruprecht J.J., Hellawell A.M., Harding M., Crichton P.G., McCoy A.J., Kunji E.R. Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism. Proc. Natl. Acad. Sci. USA. 2014;111:E426–E434. doi: 10.1073/pnas.1320692111. PubMed DOI PMC

Mavridou V., King M.S., Tavoulari S., Ruprecht J.J., Palmer S.M., Kunji E.R.S. Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle. Nat. Commun. 2022;13:3585. doi: 10.1038/s41467-022-31366-5. PubMed DOI PMC

Skulj S., Vazdar M. Calculation of apparent pKa values of saturated fatty acids with different lengths in DOPC phospholipid bilayers. Phys. Chem. Chem. Phys. 2019;21:10052–10060. doi: 10.1039/C9CP01204D. PubMed DOI

Hedger G., Rouse S.L., Domanski J., Chavent M., Koldso H., Sansom M.S. Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes. Biochemistry. 2016;55:6238–6249. doi: 10.1021/acs.biochem.6b00751. PubMed DOI PMC

Senoo N., Chinthapalli D.K., Baile M.G., Golla V.K., Saha B., Ogunbona O.B., Saba J.A., Munteanu T., Valdez Y., Whited K., et al. Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant. bioRxiv. 2023 doi: 10.1101/2023.05.05.539595. DOI

Beyer K., Klingenberg M. ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance. Biochemistry. 1985;24:3821–3826. doi: 10.1021/bi00336a001. PubMed DOI

Kunji E.R., Robinson A.J. Coupling of proton and substrate translocation in the transport cycle of mitochondrial carriers. Curr. Opin. Struct. Biol. 2010;20:440–447. doi: 10.1016/j.sbi.2010.06.004. PubMed DOI

Zoonens M., Comer J., Masscheleyn S., Pebay-Peyroula E., Chipot C., Miroux B., Dehez F. Dangerous liaisons between detergents and membrane proteins. The case of mitochondrial uncoupling protein 2. J. Am. Chem. Soc. 2013;135:15174–15182. doi: 10.1021/ja407424v. PubMed DOI

Chipot C., Dehez F., Schnell J.R., Zitzmann N., Pebay-Peyroula E., Catoire L.J., Miroux B., Kunji E.R.S., Veglia G., Cross T.A., et al. Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies. Chem. Rev. 2018;118:3559–3607. doi: 10.1021/acs.chemrev.7b00570. PubMed DOI PMC

Phelps A., Wohlrab H. Mitochondrial phosphate transport. The Saccharomyces cerevisiae (threonine 43 to cysteine) mutant protein explicitly identifies transport with genomic sequence. J. Biol. Chem. 1991;266:19882–19885. doi: 10.1016/S0021-9258(18)54864-X. PubMed DOI

Cavero S., Vozza A., del Arco A., Palmieri L., Villa A., Blanco E., Runswick M.J., Walker J.E., Cerdan S., Palmieri F., et al. Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae. Mol. Microbiol. 2003;50:1257–1269. doi: 10.1046/j.1365-2958.2003.03742.x. PubMed DOI

Wojtczak L., Wieckowski M.R., Schonfeld P. Protonophoric activity of fatty acid analogs and derivatives in the inner mitochondrial membrane: A further argument for the fatty acid cycling model. Arch. Biochem. Biophys. 1998;357:76–84. doi: 10.1006/abbi.1998.0777. PubMed DOI

Shimabukuro M., Zhou Y.T., Levi M., Unger R.H. Fatty acid-induced beta cell apoptosis: A link between obesity and diabetes. Proc. Natl. Acad. Sci. USA. 1998;95:2498–2502. doi: 10.1073/pnas.95.5.2498. PubMed DOI PMC

Kenno K.A., Severson D.L. Lipolysis in isolated myocardial cells from diabetic rat hearts. Am. J. Physiol. 1985;249:H1024–H1030. doi: 10.1152/ajpheart.1985.249.5.H1024. PubMed DOI

Van der Vusse G.J., Glatz J.F.C., Van Nieuwenhoven F.A., Reneman R.S., Bassingthwaighte J.B. Transport of long-chain fatty acids across the muscular endothelium. Skelet. Muscle Metab. Exerc. Diabetes. 1998;441:181–191. PubMed PMC

Vik-Mo H., Mjos O.D. Influence of free fatty acids on myocardial oxygen consumption and ischemic injury. Am. J. Cardiol. 1981;48:361–365. doi: 10.1016/0002-9149(81)90621-4. PubMed DOI

Takeuchi Y., Morii H., Tamura M., Hayaishi O., Watanabe Y. A possible mechanism of mitochondrial dysfunction during cerebral ischemia: Inhibition of mitochondrial respiration activity by arachidonic acid. Arch. Biochem. Biophys. 1991;289:33–38. doi: 10.1016/0003-9861(91)90438-O. PubMed DOI

Berardi M.J., Chou J.J. Fatty acid flippase activity of UCP2 is essential for its proton transport in mitochondria. Cell Metab. 2014;20:541–552. doi: 10.1016/j.cmet.2014.07.004. PubMed DOI PMC

Skulj S., Brkljaca Z., Kreiter J., Pohl E.E., Vazdar M. Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2. Int. J. Mol. Sci. 2021;22:1214. doi: 10.3390/ijms22031214. PubMed DOI PMC

Bertholet A.M., Kirichok Y. The Mechanism FA-Dependent H(+) Transport by UCP1. Brown Adipose Tissue. 2019;251:143–159. doi: 10.1007/164_2018_138. PubMed DOI

Pashkovskaya A.A., Vazdar M., Zimmermann L., Jovanovic O., Pohl P., Pohl E.E. Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface. Biophys. J. 2018;114:2142–2151. doi: 10.1016/j.bpj.2018.04.011. PubMed DOI PMC

Simard J.R., Pillai B.K., Hamilton J.A. Fatty acid flip-flop in a model membrane is faster than desorption into the aqueous phase. Biochemistry. 2008;47:9081–9089. doi: 10.1021/bi800697q. PubMed DOI

Malvezzi M., Andra K.K., Pandey K., Lee B.C., Falzone M.E., Brown A., Iqbal R., Menon A.K., Accardi A. Out-of-the-groove transport of lipids by TMEM16 and GPCR scramblases. Proc. Natl. Acad. Sci. USA. 2018;115:E7033–E7042. doi: 10.1073/pnas.1806721115. PubMed DOI PMC

Brunner J.D., Lim N.K., Schenck S., Duerst A., Dutzler R. X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature. 2014;516:207–212. doi: 10.1038/nature13984. PubMed DOI

Vork M.M., Glatz J.F.C., Vandervusse G.J. On the Mechanism of Long-Chain Fatty-Acid Transport in Cardiomyocytes as Facilitated by Cytoplasmic Fatty Acid-Binding Protein. J. Theor. Biol. 1993;160:207–222. doi: 10.1006/jtbi.1993.1014. PubMed DOI

Richieri G.V., Ogata R.T., Kleinfeld A.M. Equilibrium constants for the binding of fatty acids with fatty acid-binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB. J. Biol. Chem. 1994;269:23918–23930. doi: 10.1016/S0021-9258(19)51026-2. PubMed DOI

Pomorski T., Menon A.K. Lipid flippases and their biological functions. Cell. Mol. Life Sci. 2006;63:2908–2921. doi: 10.1007/s00018-006-6167-7. PubMed DOI PMC

Bevers E.M., Williamson P.L. Phospholipid scramblase: An update. FEBS Lett. 2010;584:2724–2730. doi: 10.1016/j.febslet.2010.03.020. PubMed DOI

Bartos L., Kabelka I., Vacha R. Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins. Biophys. J. 2021;120:2296–2305. doi: 10.1016/j.bpj.2021.04.005. PubMed DOI PMC

Suzuki J., Umeda M., Sims P.J., Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 2010;468:834–838. doi: 10.1038/nature09583. PubMed DOI

Neculai D., Schwake M., Ravichandran M., Zunke F., Collins R.F., Peters J., Neculai M., Plumb J., Loppnau P., Pizarro J.C., et al. Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36. Nature. 2013;504:172–176. doi: 10.1038/nature12684. PubMed DOI

Ruprecht J.J., King M.S., Zogg T., Aleksandrova A.A., Pardon E., Crichton P.G., Steyaert J., Kunji E.R.S. The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier. Cell. 2019;176:435–447 e415. doi: 10.1016/j.cell.2018.11.025. PubMed DOI PMC

Ballesteros A., Swartz K.J. Lipids surf the groove in scramblases. Proc. Natl. Acad. Sci. USA. 2018;115:7648–7650. doi: 10.1073/pnas.1809472115. PubMed DOI PMC

Kunji E.R., Robinson A.J. The conserved substrate binding site of mitochondrial carriers. Biochim. Biophys. Acta. 2006;1757:1237–1248. doi: 10.1016/j.bbabio.2006.03.021. PubMed DOI

Robinson A.J., Kunji E.R.S. Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proc. Natl. Acad. Sci. USA. 2006;103:2617–2622. doi: 10.1073/pnas.0509994103. PubMed DOI PMC

Dehez F., Pebay-Peyroula E., Chipot C. Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel. J. Am. Chem. Soc. 2008;130:12725–12733. doi: 10.1021/ja8033087. PubMed DOI

Robinson A.J., Overy C., Kunji E.R. The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc. Natl. Acad. Sci. USA. 2008;105:17766–17771. doi: 10.1073/pnas.0809580105. PubMed DOI PMC

Ruprecht J.J., Kunji E.R. Structural changes in the transport cycle of the mitochondrial ADP/ATP carrier. Curr. Opin. Struct. Biol. 2019;57:135–144. doi: 10.1016/j.sbi.2019.03.029. PubMed DOI PMC

Fedorenko A., Lishko P.V., Kirichok Y. Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell. 2012;151:400–413. doi: 10.1016/j.cell.2012.09.010. PubMed DOI PMC

Palmieri L., Agrimi G., Runswick M.J., Fearnley I.M., Palmieri F., Walker J.E. Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate. J. Biol. Chem. 2001;276:1916–1922. doi: 10.1074/jbc.M004332200. PubMed DOI

Wulf R., Kaltstein A., Klingenberg M. H+ and cation movements associated with ADP, ATP transport in mitochondria. Eur. J. Biochem. 1978;82:585–592. doi: 10.1111/j.1432-1033.1978.tb12054.x. PubMed DOI

LaNoue K., Mizani S.M., Klingenberg M. Electrical imbalance of adenine nucleotide transport across the mitochondrial membrane. J. Biol. Chem. 1978;253:191–198. doi: 10.1016/S0021-9258(17)38287-X. PubMed DOI

Ruprecht J.J., Kunji E.R.S. The SLC25 Mitochondrial Carrier Family: Structure and Mechanism. Trends Biochem. Sci. 2020;45:244–258. doi: 10.1016/j.tibs.2019.11.001. PubMed DOI PMC

Palmieri F. The mitochondrial transporter family SLC25: Identification, properties and physiopathology. Mol. Aspects Med. 2013;34:465–484. doi: 10.1016/j.mam.2012.05.005. PubMed DOI

Pietropaolo A., Pierri C.L., Palmieri F., Klingenberg M. The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes. Biochim. Biophys. Acta. 2016;1857:772–781. doi: 10.1016/j.bbabio.2016.02.006. PubMed DOI

Ardalan A., Sowlati-Hashjin S., Uwumarenogie S.O., Fish M., Mitchell J., Karttunen M., Smith M.D., Jelokhani-Niaraki M. Functional Oligomeric Forms of Uncoupling Protein 2: Strong Evidence for Asymmetry in Protein and Lipid Bilayer Systems. J. Phys. Chem. B. 2021;125:169–183. doi: 10.1021/acs.jpcb.0c09422. PubMed DOI

Ardalan A., Sowlati-Hashjin S., Oduwoye H., Uwumarenogie S.O., Karttunen M., Smith M.D., Jelokhani-Niaraki M. Biphasic Proton Transport Mechanism for Uncoupling Proteins. J. Phys. Chem. B. 2021;125:9130–9144. doi: 10.1021/acs.jpcb.1c04766. PubMed DOI

Kunji E.R., Crichton P.G. Mitochondrial carriers function as monomers. Biochim. Biophys. Acta. 2010;1797:817–831. doi: 10.1016/j.bbabio.2010.03.023. PubMed DOI

Kunji E.R.S., Ruprecht J.J. The mitochondrial ADP/ATP carrier exists and functions as a monomer. Biochem. Soc. Trans. 2020;48:1419–1432. doi: 10.1042/BST20190933. PubMed DOI PMC

Kunji E.R.S., King M.S., Ruprecht J.J., Thangaratnarajah C. The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology. Physiology. 2020;35:302–327. doi: 10.1152/physiol.00009.2020. PubMed DOI PMC

Wieckowski M.R., Wojtczak L. Involvement of the dicarboxylate carrier in the protonophoric action of long-chain fatty acids in mitochondria. Biochem. Biophys. Res. Commun. 1997;232:414–417. doi: 10.1006/bbrc.1997.6298. PubMed DOI

Samartsev V.N., Smirnov A.V., Zeldi I.P., Markova O.V., Mokhova E.N., Skulachev V.P. Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria. Biochim. Biophys. Acta. 1997;1319:251–257. doi: 10.1016/S0005-2728(96)00166-1. PubMed DOI

Webb B., Sali A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 2014;47:1–37. doi: 10.1002/0471250953.bi0506s47. PubMed DOI

Jo S., Lim J.B., Klauda J.B., Im W. CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes. Biophys. J. 2009;97:50–58. doi: 10.1016/j.bpj.2009.04.013. PubMed DOI PMC

Wu E.L., Cheng X., Jo S., Rui H., Song K.C., Davila-Contreras E.M., Qi Y., Lee J., Monje-Galvan V., Venable R.M., et al. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J. Comput. Chem. 2014;35:1997–2004. doi: 10.1002/jcc.23702. PubMed DOI PMC

Lee J., Cheng X., Swails J.M., Yeom M.S., Eastman P.K., Lemkul J.A., Wei S., Buckner J., Jeong J.C., Qi Y., et al. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 2016;12:405–413. doi: 10.1021/acs.jctc.5b00935. PubMed DOI PMC

Jo S., Kim T., Im W. Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS ONE. 2007;2:e880. doi: 10.1371/journal.pone.0000880. PubMed DOI PMC

Nosé S. A molecular dynamics method for simulations in the canonical ensemble. Mol. Phys. 1984;52:255–268. doi: 10.1080/00268978400101201. DOI

Parrinello M., Rahman A. Polymorphic Transitions in Single-Crystals—A New Molecular-Dynamics Method. J. Appl. Physiol. 1981;52:7182–7190. doi: 10.1063/1.328693. DOI

Essmann U., Perera L., Berkowitz M.L., Darden T., Lee H., Pedersen L.G. A smooth particle mesh Ewald method. J. Chem. Phys. 1995;103:8577–8593. doi: 10.1063/1.470117. DOI

Huang J., Rauscher S., Nawrocki G., Ran T., Feig M., de Groot B.L., Grubmuller H., MacKerell A.D., Jr. CHARMM36m: An improved force field for folded and intrinsically disordered proteins. Nat. Methods. 2017;14:71–73. doi: 10.1038/nmeth.4067. PubMed DOI PMC

Aksimentiev A., Schulten K. Imaging alpha-hemolysin with molecular dynamics: Ionic conductance, osmotic permeability, and the electrostatic potential map. Biophys. J. 2005;88:3745–3761. doi: 10.1529/biophysj.104.058727. PubMed DOI PMC

Batcho P.F., Case D.A., Schlick T. Optimized particle-mesh Ewald/multiple-time step integration for molecular dynamics simulations. J. Chem. Phys. 2001;115:4003–4018. doi: 10.1063/1.1389854. DOI

Abraham M.J., Murtola T., Schulz R., Páll S., Smith J.C., Hess B., Lindahl E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. doi: 10.1016/j.softx.2015.06.001. DOI

Humphrey W., Dalke A., Schulten K. VMD: Visual molecular dynamics. J. Mol. Graph. 1996;14:33–38. doi: 10.1016/0263-7855(96)00018-5. PubMed DOI

Exploring the proton transport mechanism of the mitochondrial ADP/ATP carrier: FA-cycling hypothesis and beyond