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Concerns with yeast mitochondrial ADP/ATP carrier’s integrity in DPC

Nature Structural & Molecular Biology volume 25, pages 747–749 (2018)Cite this article

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A Publisher Correction to this article was published on 14 September 2018

To the Editor—Herein we wish to note our objections to “Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion” by Brüschweiler et al.1. The subject of this article is the yeast mitochondrial ADP/ATP carrier AAC3, which we have studied in great detail ourselves. In particular, we have solved its structure by electron2 and X-ray3 crystallography and have studied its interactions with the specific inhibitors atractyloside (ATR) and carboxyatractyloside (CATR) by single-molecule force spectroscopy4. In ref. 1, the authors claim that AAC3 can be refolded to homogeneity from inclusion bodies produced in Escherichia coli using the detergent dodecyl phosphocholine (DPC), better known as Foscholine-12, and that AAC3 is maintained in a folded and active state for the duration of isothermal titration calorimetry (ITC) and NMR experiments. However, in our hands, the presence of DPC leads to immediate loss of tertiary structure and inactivation of AAC3 when the protein is isolated from the inner membrane of mitochondria, where it is folded and active, as shown by functional complementation2,3.

We also note that the K d of ADP binding determined by NMR (500 μM)1 is ~85-fold higher than the published consensus values for the carrier in the mitochondrial membrane and ~25-fold higher than for the solubilized carrier (Supplementary Table 2). Residues that were assigned to have chemical shift perturbations induced by ADP are largely on the matrix side of the carrier, far away from the consensus binding site in the central cavity9,10,11,12,13,14. AAC3 has an isoelectric point of 9.82, meaning that it is highly positively charged, whereas CATR and the substrates ADP and ATP are negatively charged molecules at neutral pH. The chemical shift perturbations could represent non-specific interactions of CATR or ADP with AAC3 promoted by the high concentrations and temperatures used in these NMR experiments. We are also concerned about the validity of the dynamics studies measured by NMR relaxation dispersion, as CATR binding should lock the protein in a non-dynamic, aborted state, which is why we could solve its structure by crystallography3. We also note that the observed dynamics in the presence of substrate do not provide a plausible structural mechanism for transport.

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Fig. 1: CATR binding and stability of the yeast mitochondrial ADP/ATP carrier AAC3 in dodecyl maltoside and dodecyl phosphocholine.

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Acknowledgements

We would like to acknowledge support by the intramural programme MC_UU_00015/1 of the Medical Research Council, UK.

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Authors and Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK

    Martin S. King, Paul G. Crichton, Jonathan J. Ruprecht & Edmund R. S. Kunji

  2. Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK

    Paul G. Crichton

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  1. Martin S. King
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Correspondence to Edmund R. S. Kunji.

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King, M.S., Crichton, P.G., Ruprecht, J.J. et al. Concerns with yeast mitochondrial ADP/ATP carrier’s integrity in DPC . Nat Struct Mol Biol 25, 747–749 (2018). https://doi.org/10.1038/s41594-018-0125-6

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