Abstract

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

Supplementary key wordsAbbreviations: AAC (ADP/ATP carrier), CATR (carboxyatractyloside), CL (cardiolipin), IMM (the inner mitochondrial membrane), MCF (the mitochondrial carrier family), MD (molecular dynamics), OXPHOS (Oxidative phosphorylation), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), RMSF (root mean square fluctuations), TM (transmembrane)The inner mitochondrial membrane (IMM) exhibits a variety of unique properties: forming cristae leads to very large surface area; proteins are densely packed within it; and the membrane contains featured phospholipids, cardiolipins (CLs). IMM is crowded with membrane proteins involved in energy production and substrate transportation, including oxidative phosphorylation (OXPHOS) complexes and mitochondrial carriers. Some studies estimate the protein/lipid ratio reaches to as high as 3:1 (1Hallermayer G. Neupert W. Lipid composition of mitochondrial outer and inner membranes of Neurospora crassa., 2Nicholls D.G. Ferguson S.J. , 3Formation and regulation of mitochondrial membranes.), in contrast to roughly 1:1 in cell membranes (4The Cell: A Molecular Approach.). Because IMM is densely packed with OXPHOS complexes, there is little potential to improve OXPHOS capacity through increasing density of OXPHOS proteins. One important strategy is to increase the surface area of IMM through forming highly folded cristae structure. It is reported that CLs might play an important role on cristae biogenesis of mitochondria (5Cardiolipin and mitochondrial cristae organization.). CLs are unique lipids preserved during evolution from aerobic bacteria to mitochondria, and in mitochondria, CLs are mainly distributed within the inner leaflet of IMM where they are generated (6Baile M.G. Lu Y.-W. Claypool S.M. The topology and regulation of cardiolipin biosynthesis and remodeling in yeast.). CLs roughly account for 10%–20% of total phospholipids in IMM (, 8Dowhan W. Bogdanov M. Mileykovskaya E. Functional Roles of Lipids in Membranes.). The structure of CL is featured with a small hydroxyl head group and four hydrophobic acyl tails (Fig. 1A), and it usually carries two negative net charges in physiological conditions. Its special conical shape makes CLs have potential to relax curvature strain in cristae. Moreover, CLs have been reported to stabilize and regulate the function of a bunch of mitochondrial proteins such as cytochrome c, ADP/ATP carrier (AAC), and ATPase (9Ren M. Phoon C.K.L. Schlame M. Metabolism and function of mitochondrial cardiolipin., 10Duncan A.L. Robinson A.J. Walker J.E. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases., 11Cardiolipin and mitochondrial carriers., 12Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin., 13Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. et al.Cardiolipin stabilizes respiratory chain supercomplexes., 14Cardiolipin, a critical determinant of mitochondrial carrier protein assembly and function., 15Senoo N. Kandasamy S. Ogunbona O.B. Baile M.G. Lu Y. Claypool S.M. Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast.).

Fig. 1Structures of cardiolipin and the ADP/ATP carrier. A: Chemical and tertiary structure of CL. B: The tripartite secondary structure of AAC. Domains are shown in distinct colors with domain 1 in yellow, domain 2 in orange, and domain 3 in red. C: Side view (top) and matrix view (bottom) of the tertiary structure of AAC with three specifically bound CLs. CLs are highlighted in blue stick-ball model. AAC, ADP/ATP carrier; CL, cardiolipin.

AAC is the most abundant protein in IMM and plays a central role in OXPHOS. It is responsible for importing ADP into the mitochondrial matrix and exporting ATP out of the mitochondria for living cells in a 1:1 exchange. The exchange of ADP and ATP across the IMM is completed through AAC alternating from the cytosol-open (c-) or ADP-waiting conformation to the matrix-open (m-) or ATP-waiting conformation via the transition state (16Pietropaolo A. Pierri C.L. Palmieri F. Klingenberg M. The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes.). Highly specific inhibitors including atractyloside and carboxyatractyloside (CATR) help lock AAC at c-state (17The ADP and ATP transport in mitochondria and its carrier., 18Pebay-Peyroula E. Dahout-Gonzalez C. Kahn R. Trézéguet V. Lauquin G.J. Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside., 19Todisco S. Di Noia M.A. Onofrio A. Parisi G. Punzi G. Redavid G. et al.Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.), while bongkrekic acid helps fix AAC at m-state (17The ADP and ATP transport in mitochondria and its carrier., 19Todisco S. Di Noia M.A. Onofrio A. Parisi G. Punzi G. Redavid G. et al.Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays., 20Ruprecht J.J. King M.S. Zögg T. Aleksandrova A.A. Pardon E. Crichton P.G. et al.The Molecular mechanism of transport by the mitochondrial ADP/ATP carrier.). Benefitting from the high expression levels and the presence of state-specific inhibitors, AAC has long been paradigm of the big mitochondrial carrier family (MCF) (21Boxer D.H. Feckl J. Klingenberg M. Identity between the major protein located at the outer face of the inner mitochondrial membrane and carboxyatractylate binding protein., 22Palmieri F. Pierri C.L. De Grassi A. Nunes-Nesi A. Fernie A.R. Evolution, structure and function of mitochondrial carriers: a review with new insights.), and this family functions to mediate a variety of metabolites across IMM. Currently 53 MCF members have been identified in human genome, and they form the biggest solute carrier (SLC) subfamily which is also referred to as SLC25. Like other members of MCF, AAC is featured with three homologous repeats (Fig. 1B), each containing about 100 amino acids with highly conserved MCF motif Px[DE]xx[KR]xRxQ-(matrix loop and matrix helix)-[DE]Gxxxx[YWF][KR]G near the matrix side (23Internal sequence repeats and the path of polypeptide in mitochondrial ADP/ATP translocase., 24Nelson D.R. Felix C.M. Swanson J.M. Highly conserved charge-pair networks in the mitochondrial carrier family., 25Aquila H. Misra D. Eulitz M. Klingenberg M. Complete amino acid sequence of the ADP/ATP carrier from beef heart mitochondria., 26Walker J.E. Runswick M.J. The mitochondrial transport protein superfamily., 27Mitochondrial carrier proteins., 28Pierri C.L. Palmieri F. De Grassi A. Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers., 29Yi Q. Li Q. Yao S. Chen Y. Guan M.-X. Cang X. Molecular dynamics simulations on apo ADP/ATP carrier shed new lights on the featured motif of the mitochondrial carriers.), with the cysteine-containing matrix short helices hosting the sequence motif -50-QYKGxxDCxRK-60 (sequence numbering referred to the bovine AAC1 sequence) (28Pierri C.L. Palmieri F. De Grassi A. Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers.).Although CL is not absolutely required for AAC function (14Cardiolipin, a critical determinant of mitochondrial carrier protein assembly and function.), evidences have shown that CL can effectively stabilize AAC in purification and reconstitution (18Pebay-Peyroula E. Dahout-Gonzalez C. Kahn R. Trézéguet V. Lauquin G.J. Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside., 20Ruprecht J.J. King M.S. Zögg T. Aleksandrova A.A. Pardon E. Crichton P.G. et al.The Molecular mechanism of transport by the mitochondrial ADP/ATP carrier., 30Ruprecht 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.), act as a transport activator (31Hoffmann B. Stöckl A. Schlame M. Beyer K. Klingenberg M. The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants., 32Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. et al.Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.), affect conformational preference of AAC (15Senoo N. Kandasamy S. Ogunbona O.B. Baile M.G. Lu Y. Claypool S.M. Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast.) and regulate the c/m conformational transition (30Ruprecht 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., 33Specific cardiolipin binding interferes with labeling of sulfhydryl residues in the adenosine diphosphate/adenosine triphosphate carrier protein from beef heart mitochondria.). Tight binding between AAC and CL was first identified using 31P-NMR in 1985 (34ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by phosphorus-31 nuclear magnetic resonance.) and was further confirmed by crystal structures of AAC in both c- and m-states (18Pebay-Peyroula E. Dahout-Gonzalez C. Kahn R. Trézéguet V. Lauquin G.J. Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside., 20Ruprecht J.J. King M.S. Zögg T. Aleksandrova A.A. Pardon E. Crichton P.G. et al.The Molecular mechanism of transport by the mitochondrial ADP/ATP carrier., 30Ruprecht 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., 35Nury H. Dahout-Gonzalez C. Trézéguet V. Lauquin G. Brandolin G. Pebay-Peyroula E. Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.). In these structures, six transmembrane (TM) α helices form a bundle surrounding a cone-shaped cavity opening toward the intermembrane space in c-state and toward the matrix in m-state. Each odd-numbered helix is connected to the following even-numbered helix at the matrix side through a flexible loop, a short amphipathic α helix and a linker helix, which forms the basic structural scaffold for one repeat domain of AAC (Fig. 1B, C). In c-state structure, three repeated homologous domains of AAC show three-fold pseudosymmetry, and at each domain–domain interface, there is one specifically bound CL (Fig. 1C). Based on the static crystal structures, it was proposed that CLs bind with the exposed positively charged amide groups and helix dipoles at the N-terminal ends of even-numbered helices and short matrix helices (30Ruprecht 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.). However, molecular dynamics (MD) simulations have shown that positively charged and polar residues also play an important role in binding with CLs (36Mao X. Yao S. Yi Q. Xu Z.-M. Cang X. Function-related asymmetry of the specific cardiolipin binding sites on the mitochondrial ADP/ATP carrier., 37Pasquadibisceglie A. Polticelli F. Structural determinants of ligands recognition by the human mitochondrial basic amino acids transporter SLC25A29. Insights from molecular dynamics simulations of the c-state.), and this result is in agreement with previous experiments (11Cardiolipin and mitochondrial carriers., 38Bogner W. Aquila H. Klingenberg M. The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate., 39Characterization of the novel cardiolipin binding regions identified on the protease and lipid activated PKC-related kinase 1.). The difference from crystal structures and MD simulations highlights the importance of introducing structural dynamics in exploring lipid–protein interactions, which is also supported by studies from other groups (10Duncan A.L. Robinson A.J. Walker J.E. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases., 40Muller M.P. Jiang T. Sun C. Lihan M. Pant S. Mahinthichaichan P. et al.Characterization of Lipid–Protein Interactions and Lipid-Mediated Modulation of Membrane Protein Function through Molecular Simulation., 41Hedger G. Rouse S.L. Domański J. Chavent M. Koldsø H. Sansom M.S.P. Lipid-loving ANTs: molecular simulations of cardiolipin interactions and the organization of the adenine nucleotide translocase in model mitochondrial membranes., 42Duncan A.L. Ruprecht J.J. Kunji E. Robinson A.J. Cardiolipin dynamics and binding to conserved residues in the mitochondrial ADP/ATP carrier., 43Cang X. Du Y. Mao Y. Wang Y. Yang H. Jiang H. Mapping the Functional binding sites of cholesterol in β2-adrenergic receptor by long-time molecular dynamics simulations., 44Hanske J. Toffey J.R. Morenz A.M. Bonilla A.J. Schiavoni K.H. Pletneva E.V. Conformational properties of cardiolipin-bound cytochrome c., 45Cang X. Yang L. Yang J. Luo C. Zheng M. Yu K. et al.Cholesterol-β1AR interaction versus cholesterol-β2AR interaction.). Due to varied distributions of the positively charged and polar residues in the three specific CL binding sites, our previous work reported that the three sites are highly asymmetric, and the asymmetric CL binding behavior at the three sites is quite consistent with the asymmetry observed in the matrix side of the m-state structure (20Ruprecht J.J. King M.S. Zögg T. Aleksandrova A.A. Pardon E. Crichton P.G. et al.The Molecular mechanism of transport by the mitochondrial ADP/ATP carrier.). However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive.

In the current work, three 3-μs all-atom MD simulations on c-state apo AAC with CLs (CL-1, 2, 3) are compared to the three 3-μs simulations without CLs (PC-1, 2, 3). We analyzed the impact of CLs on both macroscopic parameters such as pocket volume of AAC and microscopic parameters such as interactions among MCF motif residues. Significantly, we have revealed a strictly conserved stacking structure R30:R71:R151 at the domain 1-2 interface, and we speculate that this stacking structure functions as a structural switch under regulation of CLs.

Materials and methodsInitial preparation of the simulation systemsThe starting protein coordinates were obtained from the CATR-inhibited crystal structure of AAC (PDB entry: 1okc) (18Pebay-Peyroula E. Dahout-Gonzalez C. Kahn R. Trézéguet V. Lauquin G.J. Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.). The missing residues in the crystal structure of AAC were added using iTasser (46Yang J. Yan R. Roy A. Xu D. Poisson J. Zhang Y. The I-TASSER Suite: protein structure and function prediction.). The termini residues of AAC were manipulated into a charged state by protonating the N terminus and deprotonating the C terminus. A pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane and a mixed membrane composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine, and CL were built with the Membrane Builder modules of VMD (47Humphrey W. Dalke A. Schulten K. VMD – Visual molecular dynamics.) and CHARMM-GUI (48Jo S. Kim T. Iyer V.G. Im W. CHARMM-GUI: a web-based graphical user interface for CHARMM.