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Dynamics and interactions of AAC3 in DPC are not functionally relevant

Nature Structural & Molecular Biology volume 25, pages 745–747 (2018)Cite this article

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To the Editor – A recent study by Brüschweiler et al.1 used solution-state NMR spectroscopy to examine the interactions and dynamics of the yeast mitochondrial inner membrane ADP/ATP carrier (yAAC3). Crystal structures of different AACs, including yAAC3, in a conformation locked with a strong inhibitor (CATR) had been determined before. This putative ‘c-state’2,3 is believed to represent an extreme conformation of an alternating-access mechanism, which involves a further and yet-elusive second state termed the ‘m-state’. Characterizing the dynamics between these states is of paramount importance to understanding the transport mechanism. Brüschweiler et al.1 refolded yAAC3 from inclusion bodies in the detergent dodecylphosphocholine (DPC) and observed micro- to millisecond motion in part of yAAC3 using CPMG NMR experiments. The authors propose that these asymmetrically distributed dynamics, involving residues located in three of six helices (α1, α2 and α6), correspond to excursions from the c-state, which they believe is the predominant state in their sample, to an alternative state populated to 2%. They further propose that this transiently populated state might correspond to, or at least be similar to, the long-sought m-state. Support for the importance of the sparse state in functional turnover comes from their finding that dynamics are quantitatively different when the substrate (ADP) or inhibitor (CATR) is present. As discussed below, we disagree with their interpretations. Specifically, we believe that (i) the sample was not in a functionally relevant folded state, (ii) the dynamics fits were not correctly performed and the dynamics parameters are quantitatively incorrect, and (iii) the substrate or inhibitor has negligible effects on dynamics in their sample, thus challenging their main conclusions.

A number of studies have demonstrated the denaturing effects of DPC on AAC and related mitochondrial carriers4–8. The authors chose to study binding of CATR and ADP as criteria to assess whether their yAAC3 sample in DPC was functional. They report a dissociation constant (K d) for CATR binding of about 26 µM (using isothermal titration calorimetry (ITC)) to 150 µM (by NMR). The apparent agreement with one particular literature value9 of 192 µM would suggest that yAAC3 is functional in DPC. However, the originally reported value was a typographical error and has since been corrected to 192 nM10, in agreement with many previously reported low-nanomolar K d values in membranes and detergents7,12,13,14. The CATR affinity in DPC is therefore about three orders of magnitude lower than the one for a functional protein. This finding by itself strongly suggests that the sample is in a non-native state.

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Fig. 1: Solute-induced NMR chemical shift perturbations, interactions and millisecond motions of yAAC3 in DPC.

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Acknowledgements

This work was supported by the European Research Council (ERC-StG-311318).

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Authors and Affiliations

  1. Univ. Grenoble Alpes, CEA, CNRS, Institute for Structural Biology (IBS), Grenoble, France

    Vilius Kurauskas, Audrey Hessel, Beate Bersch & Paul Schanda

  2. LPCT, UMR 7019, Université de Lorraine, CNRS, Vandoeuvre-les-Nancy, France

    François Dehez & Christophe Chipot

  3. Laboratoire International Associé CNRS and University of Illinois at Urbana–Champaign, Vandoeuvre-les-Nancy, France

    François Dehez & Christophe Chipot

  4. Department of Physics, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Christophe Chipot

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  1. Vilius Kurauskas
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Correspondence to Paul Schanda.

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Kurauskas, V., Hessel, A., Dehez, F. et al. Dynamics and interactions of AAC3 in DPC are not functionally relevant. Nat Struct Mol Biol 25, 745–747 (2018). https://doi.org/10.1038/s41594-018-0127-4

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