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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    • "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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    • "The second approach used molecular dynamics simulations to identify residues of the bovine mitochondrial ADP/ATP carrier that are involved in the trajectory and binding of ADP in the cavity. ADP bound with a minimum of the free energy to the binding site defined above, revealing an aromatic stacking arrangement of the adenine moiety with Y186 and an ionic interaction of the b-phosphate of ADP with an arginine, which comes through the centre of the matrix salt bridge network (Dehez et al. 2008, Wang and Tajkhorshid 2008). From a sequence perspective, it was clear that the most important interactions of the carriers with the substrate were electrostatic in nature (Kunji and Robinson 2006), but this notion has been refined by calculating electrostatic potentials (Wang and Tajkhorshid 2008) and by looking at the effects of chlorine ions on binding (Krammer et al. 2009). "
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    ABSTRACT: Abstract Mitochondrial carriers transport inorganic ions, nucleotides, amino acids, keto acids and cofactors across the mitochondrial inner membrane. Structurally they consist of three domains, each containing two transmembrane α-helices linked by a short α-helix and loop. The substrate binds to three major contact points in the central cavity. The class of substrate (e.g., adenine nucleotides) is determined by contact point II on transmembrane α-helix H4 and the type of substrate within the class (e.g., ADP, coenzyme A) by contact point I in H2, whereas contact point III on H6 is most usually a positively charged residue, irrespective of the type or class. Two salt bridge networks, consisting of conserved and symmetric residues, are located on the matrix and cytoplasmic side of the cavity. These residues are part of the gates that are involved in opening and closing of the carrier during the transport cycle, exposing the central substrate binding site to either side of the membrane in an alternating way. Here we revisit the plethora of mutagenesis data that have been collected over the last two decades to see if the residues in the proposed binding site and salt bridge networks are indeed important for function. The analysis shows that the major contact points of the substrate binding site are indeed crucial for function and in defining the specificity. The matrix salt bridge network is more critical for function than the cytoplasmic salt bridge network in agreement with its central position, but neither is likely to be involved in substrate recognition directly.
    Molecular Membrane Biology 03/2013; 30(2):149-159. DOI:10.3109/09687688.2012.737936 · 1.69 Impact Factor
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François Dehez