Substrate Modulated Dynamics of the ADP/ATP Transporter Revealed by NMR Relaxation Dispersion

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Abstract

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters that have 2-fold direct or inverted quasi-symmetry, AAC has the apparent 3-fold rotational symmetry. Further, its transport rate is fast for transporters that carry large solutes. Here, we perform comprehensive NMR relaxation dispersion measurements for the yeast AAC carrier 3, which provide residue-specific information on the protein conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a lowly populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP significantly increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that while the transporter catalyzes the translocation of substrate, the substrate also facilitates interconversion between alternating states that may be relevant to the transport function.

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Most cited references 34

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The dynamic process of β(2)-adrenergic receptor activation.

Rie Nygaard, Yaozhong Zou, Ron Dror … (2013)

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

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Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

Eva Pebay-Peyroula, Cécile Dahout-Gonzalez, Richard Kahn … (2003)

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation.

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The ADP and ATP transport in mitochondria and its carrier.

Martin Klingenberg (2008)

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP/ATP transport in mitochondria is presented. The obvious need for an "old fashioned" review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more years ago, of unravelling the role of mitochondrial compartments and of the two membranes, the sequence of steps of how ATP arrives in the cytosol became a major issue. When the dust settled, a picture emerged where ATP is exported across the inner membrane in a 1:1 exchange against ADP and where the selection of ATP versus ADP is controlled by the high membrane potential at the inner membrane, thus uplifting the free energy of ATP in the cytosol over the mitochondrial matrix. Thus the disparate energy and redox states of the two major compartments are bridged by two membrane potential responsive carriers to enable their symbiosis in the eukaryotic cell. The advance to the molecular level by studying the binding of nucleotides and inhibitors was facilitated by the high level of carrier (AAC) binding sites in the mitochondrial membrane. A striking flexibility of nucleotide binding uncovered the reorientation of carrier sites between outer and inner face, assisted by the side specific high affinity inhibitors. The evidence of a single carrier site versus separate sites for substrate and inhibitors was expounded. In an ideal setting principles of transport catalysis were elucidated. The isolation of intact AAC as a first for any transporter enabled the reconstitution of transport for unravelling, independently of mitochondrial complications, the factors controlling the ADP/ATP exchange. Electrical currents measured with the reconstituted AAC demonstrated electrogenic translocation and charge shift of reorienting carrier sites. Aberrant or vital para-functions of AAC in basal uncoupling and in the mitochondrial pore transition were demonstrated in mitochondria and by patch clamp with reconstituted AAC. The first amino acid sequence of AAC and of any eukaryotic carrier furnished a 6-transmembrane helix folding model, and was the basis for mapping the structure by access studies with various probes, and for demonstrating the strong conformation changes demanded by the reorientation mechanism. Mutations served to elucidate the function of residues, including the particular sensitivity of ATP versus ADP transport to deletion of critical positive charge in AAC. After resisting for decades, at last the atomic crystal structure of the stabilised CAT-AAC complex emerged supporting the predicted principle fold of the AAC but showing unexpected features relevant to mechanism. Being a snapshot of an extreme abortive "c-state" the actual mechanism still remains a conjecture.

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Journal

Journal ID (nlm-journal-id): 101186374

Journal ID (pubmed-jr-id): 31761

Journal ID (nlm-ta): Nat Struct Mol Biol

Journal ID (iso-abbrev): Nat. Struct. Mol. Biol.

Title: Nature structural & molecular biology

ISSN (Print): 1545-9993

ISSN (Electronic): 1545-9985

Publication date Nihms-submitted: 6 July 2015

Publication date (Electronic): 13 July 2015

Publication date (Print): August 2015

Publication date PMC-release: 01 February 2016

Volume: 22

Issue: 8

Pages: 636-641

Affiliations

[1 ]Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

[2 ]National Center for Protein Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

[3 ]State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

Author notes

[* ]Corresponding author: james_chou@ 123456hms.harvard.edu

[†]

The authors contributed equally.

Article

Manuscript ID: NIHMS701289

DOI: 10.1038/nsmb.3059

PMC ID: 4527935

PubMed ID: 26167881

SO-VID: 31cf11ac-ef69-4cbf-83ae-32dab77203cd

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Substrate Modulated Dynamics of the ADP/ATP Transporter Revealed by NMR Relaxation Dispersion

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Abstract

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Higher order chromatin architecture

Most cited references 34

The dynamic process of β(2)-adrenergic receptor activation.

Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

The ADP and ATP transport in mitochondria and its carrier.

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