Binding of ADP in the Mitochondrial ADP/ATP Carrier Is Driven by an Electrostatic Funnel

Equipe de dynamique des assemblages membranaires, UMR No. 7565 CNRS-UHP, Nancy Université, BP 239, 54506 Vandoeuvre-lès-Nancy cedex, France.
Journal of the American Chemical Society (Impact Factor: 12.11). 09/2008; 130(38):12725-33. DOI: 10.1021/ja8033087
Source: PubMed


The ADP/ATP carrier (AAC) is a membrane protein of paramount importance for the energy-fueling function of the mitochondria, transporting ADP from the intermembrane space to the matrix and ATP in the opposite direction. On the basis of the high-resolution, 2.2-A structure of the bovine carrier, a total of 0.53 micros of classical molecular dynamics simulations were conducted in a realistic membrane environment to decipher the early events of ADP (3-) translocation across the inner membrane of the mitochondria. Examination of apo-AAC underscores the impermeable nature of the carrier, impeding passive transport of permeants toward the matrix. The electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential forms a privileged passageway anticipated to drive the diphosphate nucleotide rapidly toward the bottom of the internal cavity. This conjecture is verified in the light of repeated, independent numerical experiments, whereby the permeant is dropped near the mouth of the mitochondrial carrier. Systematic association of ADP (3-) to the crevice of the AAC, an early event in its transport across the inner membrane, is accompanied by the formation of an intricate network of noncovalent bonds. Simulations relying on the use of an adaptive biasing force reveal for the first time that the proposed binding site corresponds to a minimum of the free energy landscape delineating the translocation of ADP (3-) in the carrier. The present work paves the way to the design of novel nucleotides and new experiments aimed at unveiling key structural features in the chronology of ADP/ATP transport across the mitochondrial membrane.

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    • "Consequently, a substantial amount of interaction energy must be involved in substrate binding too in order to lower the energy barrier sufficiently for transport to occur. The substrate binding site of the bovine mitochondrial ADP/ATP carrier has been proposed to consist of G182, I183 and Y186 for binding of the adenine moiety and R79, K22 and R279 for binding of the phosphate groups [14] [15] [16] [17] [18], which could form an aromatic stacking and three ionic interactions with ADP, consistent with this idea. By introducing mutations it was possible to increase or decrease the interaction energy of the cytoplasmic network in a systematic way (colored levels, Fig. 6). "
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    ABSTRACT: Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway.
    Biochimica et Biophysica Acta 10/2015; 1857(1). DOI:10.1016/j.bbabio.2015.09.013 · 4.66 Impact Factor
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    • "The PRE results indicate that GDP binds deep inside the hydrophilic cavity of UCP2, with the pyrophosphate group near the proline kinks (Figure 3B) (Berardi et al., 2011), which are the key structural signatures of mitochondrial carriers. This mode of nucleotide binding is consistent with the proposed ADP binding site in AAC (Dehez et al., 2008; Kunji and Robinson, 2006; Wang and Tajkhorshid, 2008); it is also consistent with the depth of ATP binding in UCP1 (i.e., near the P-kinks about midway through the cavity) measured by atomic force microscopy (Zhu et al., 2013). The NMR measurements of FA and GDP interactions with UCP2 indicate allosteric interplay between FA and GDP in which GDP binding inside the cavity can displace FA from its binding site on the outside. "
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    ABSTRACT: Modulation of cellular energy expenditure is fundamental to normal and pathological cell growth and differentiation. Mitochondria stores energy as a proton gradient across their inner membrane. Uncoupling proteins (UCPs) can dissipate the gradient to produce heat or regulate metabolite fluxes. UCP-mediated proton currents require fatty acids (FAs) and are blocked by nucleotides, but the molecular basis of these processes is unknown. We find, by nuclear magnetic resonance and functional mutagenesis, that UCP2 can bind FAs laterally through its peripheral site, and this intramembrane molecular recognition is essential for UCP2-catalyzed FA flipping across the membrane, which in turn is essential for proton translocation. The antagonist GDP binds inside the UCP2 cavity and perturbs its conformation, which can displace FA from the peripheral site as a mean of inhibiting proton currents. Our data provide a biophysical perspective of the intricate interplay of UCPs, FA, and nucleotides in determining proton fluxes in mitochondria.
    Cell Metabolism 08/2014; 20(3). DOI:10.1016/j.cmet.2014.07.004 · 17.57 Impact Factor
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    • "Nam et al. reported their discovery that electrostatic interactions accounted for the majority of the rate acceleration in the mechanism of RNA transphosphorylation in solution catalyzed by the hairpin ribozyme [38]. Moreover, the electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential was reported by Dehez et al., driving the diphosphate nucleotide rapidly toward the bottom of the internal cavity of membrane-protein mitochondrial ADP/ATP carrier by forming a privileged passageway [39]. Taking into account these findings comprehensively, we assumed that the electrostatic potential of the inhibitor also played a important role in the binding and interaction with chymase together with orbital energy and consequently influenced the inhibition effect. "
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    ABSTRACT: Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure-based pharmacophore models. One ligand-based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well.
    PLoS ONE 04/2013; 8(4):e63030. DOI:10.1371/journal.pone.0063030 · 3.23 Impact Factor
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