Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter (original) (raw)

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Electron Microscopy Data Bank

Data deposits

All three-dimensional cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6085 (TmrAB–AH5), EMD-6086 (TmrAB–BA6) and EMD-6087 (TmrAB).

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Acknowledgements

This work was supported by grants from the National Institutes of Health (R01GM098672, S10RR026814 and P50GM082250 to Y.C., 1P41CA196276-01 to C.S.C., P50GM073210 to R.M.S. and C.S.C., and R37GM024485 to R.M.S.), the University of California San Francisco Program for Breakthrough Biomedical Research (to Y.C.), and the German Research Foundation (SFB 807, SFB 902 and TA157/7 to R.T.) as well as the European Drug Initiative on Channels and Transporters (EDICT to R.T.) funded by the European Commission Seventh Framework Program.

Author information

Author notes

  1. JungMin Kim, Shenping Wu and Thomas M. Tomasiak: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA,
    JungMin Kim, Michael B. Winter, Robert M. Stroud & Charles S. Craik
  2. Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA,
    Shenping Wu, Thomas M. Tomasiak, Yaneth Robles-Colmanares, Robert M. Stroud & Yifan Cheng
  3. Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany,
    Claudia Mergel, Sebastian B. Stiller & Robert Tampé
  4. Cluster of Excellence – Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany,
    Robert Tampé

Authors

  1. JungMin Kim
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  2. Shenping Wu
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  3. Thomas M. Tomasiak
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  4. Claudia Mergel
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  5. Michael B. Winter
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  6. Sebastian B. Stiller
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  7. Yaneth Robles-Colmanares
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  8. Robert M. Stroud
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  9. Robert Tampé
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  10. Charles S. Craik
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  11. Yifan Cheng
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Contributions

J.K. identified, expressed, purified and characterized all Fabs used in this study, and generated TmrAB–Fab complexes. S.W. performed all cryo-EM experiments, including data acquisition and processing. T.M.T. and C.M. expressed and purified TmrAB, and purified TmrAB–Fab complexes. T.M.T. performed cross-linking experiments. C.M. expressed and purified TmrAB for the generation and initial screening of all Fabs. S.B.S. performed initial characterization of all Fabs. M.B.W. performed high-performance liquid chromatography (HPLC) experiments. Y.R.-C. performed mutagenesis experiments. J.K., S.W., T.M.T. and Y.C. analysed data. J.K., S.W., T.M.T., M.B.W., R.M.S., R.T., C.S.C. and Y.C. participated in discussion and wrote the manuscript.

Corresponding authors

Correspondence toRobert M. Stroud, Robert Tampé, Charles S. Craik or Yifan Cheng.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Binding characterization of Fabs against TmrAB.

a, Expression levels of Fabs used in ELISA in Fig. 1a. Expression levels were assessed by immunoblotting with anti-c-myc antibody and normalized against the highest expression level such that undiluted AD12 equals a normalized relative concentration of 1. Binding was monitored by anti-c-myc antibody. b, ELISA with purified Fabs against TmrAB. Purified Flag-tagged Fabs (Fab–Flag) were used in binding reactions. Binding was monitored by anti–Flag M2-peroxidase. Experiments were repeated twice. c, Relative affinities of the Fabs. The ELISA signal data from Extended Data Fig. 1b were fitted to a bimolecular binding equation to produce binding curves and apparent dissociation constant (_K_d) values. AH5 showed the highest affinity, followed by BA6, AD12 and AH11. DH5 and BG12 did not show significant binding. Apparent _K_d values do not yield meaningful biophysical properties because the concentration of TmrAB participating in the binding reaction is unknown and the transporter is not free in solution. However, comparison among the Fabs should be sufficient to determine their relative affinities. Unpurified DH5 showed significant binding (Extended Data Fig. 1a), whereas purified DH5 did not show significant binding; n.d., not determined. d, Negative-stain electron microscopy analysis of Fab +TmrAB mixes. Representative two-dimensional class averages include complex images that show clear Fab densities and images that do not. The two typical Fab views, the dumb-bell- and doughnut-shaped views, are indicated by yellow and red arrows respectively. Fab images indicate that Fabs are rigid and form rigid complexes. Percentages of complex particles were assessed by fractions of the numbers of images that clearly show Fab densities (# complex particles) to the total numbers of images that were included (# total particles) in two-dimensional class averages. The percentages correspond to relative affinity ranking determined by ELISA (Extended Data Fig. 1c).

Extended Data Figure 2 Purification of α-DDM-solubilized and single-particle cryo-EM of TmrAB–AH5 complex.

a, Elution profiles of TmrAB alone and TmrAB–AH5 from Superdex 200 are shown in blue and red curves respectively, showing a clear shift of the elution peak of the TmrAB–AH5 complex to a higher molecular mass position. The shifted peak corresponding to fractions A6–A8 contained TmrAB and AH5, confirmed by SDS–PAGE. Fractions B3–B5 correspond to unbound AH5 and the loading material was run for comparison. b, Raw micrograph of TmrAB–AH5 (∼185 kDa) embedded in a thin layer of vitreous ice. c, Fourier power spectrum calculated from micrograph shown in a. d, Two-dimensional class averages of the TmrAB–AH5 complex. Fab AH5 is clearly visible in many class averages. e, Initial three-dimensional reconstruction calculated from two-dimensional class averages using the common lines method implemented in SPIDER. f, Fourier shell correlation curves of TmrAB–AH5 (red), TmrAB–BA6 (purple) and TmrAB alone (blue). g, Euler angle distribution of all particles used in the final reconstruction. h, Final three-dimensional reconstruction coloured with local resolution. i, Voxel histogram corresponding to local resolution. The majority of voxels are at ∼6–7 Å resolution. Estimation of local resolution that is too close to the Nyquist (3.9 Å) may not be accurate.

Extended Data Figure 3 Selected slice views of the three-dimensional reconstruction of TmrAB–AH5.

The views are oriented in parallel with the membrane plane. The numbers of slices are marked. a, All transmembrane helices of both TmrA and TmrB are labelled. The arrow points to the extra density in the cavity. b, Two NBDs are in contact with each other. c, The C-terminal helices of TmrA and TmrB are in close contact.

Extended Data Figure 4 Single-particle cryo-EM of TmrAB–BA6 complex.

a, Raw micrograph of TmrAB–BA6 (∼185 kDa) embedded in a thin layer of vitreous ice. Images were collected on a Tecnai TF20 microscope using a scintillator-based TVIPS 8k × 8k CMOS camera. b, Fourier power spectrum calculated from the micrograph shown in a. c, Two-dimensional class averages of the TmrAB–BA6 complex. Fab BA6 is clearly visible in many class averages. d, Initial three-dimensional reconstruction of TmrAB–BA6 determined using the common lines method implemented in SPIDER. e, Two different views of the final three-dimensional reconstruction of TmrAB–BA6 filtered to a resolution of 9.4 Å. As in the three-dimensional reconstruction of TmrAB–AH5, the density of micelles is split into two halves and tilted with respect to each other. The orientation of micelle density is marked with a pair of black solid lines and the gap in the micelle density generated by the helix H4 from TmrB is marked with a pair of red dotted lines. f, Densities of TmrAB in the three-dimensional reconstructions of TmrAB–AH5 (khaki) and TmrAB–BA6 (grey mesh) overlap. Fabs AH5 and BA6 are indicated with arrows. g, An enlarged view to show the interface between TmrAB and BA6, which has a linear epitope in the NBD of TmrB.

Extended Data Figure 5 Single-particle cryo-EM of TmrAB alone without Fab.

a, Raw micrograph of TmrAB alone (∼135 kDa) embedded in a thin layer of vitreous ice. Images were collected on a Tecnai TF20 microscope using scintillator based TVIPS 8k × 8k CMOS camera. b, Fourier power spectrum calculated from micrograph shown in a. c, Two-dimensional class averages of TmrAB. d–f, Three different views of TmrAB three-dimensional reconstruction shown in different (low: grey; high: gold) contour levels. Model of TmrAB (in ribbon diagram) was docked into the density map. The orientation of micelle density is indicated with pairs of solid black lines in f and the gap in the micelle is indicated with a pair of red dotted lines. g, h, Densities of TmrAB in the three-dimensional reconstructions of TmrAB alone (transparent khaki) and in complex with AH5 (grey mesh) overlap each other.

Extended Data Figure 6 Cross correlation between TmrAB–AH5, TmrAB–BA6 and TmrAB.

Left: density map of TmrAB–AH5 is coloured according to the value of local cross-correlation values of TmrAB–AH5 with TmrAB–BA6 (upper), with TmrAB (lower). Middle: density map of TmrAB-BA6 is coloured according to the value of local cross-correlation values of TmrAB–BA6 with TmrAB–AH5 (upper), and with TmrAB (lower). Right: density map of TmrAB is coloured according to the local cross-correlation value of TmrAB with TmrAB–AH5 (upper) and with TmrAB–BA6 (lower).

Extended Data Figure 7 Atomic model of TmrAB.

a, b, Two different views of the atomic model of TmrAB, generated by flexible fitting of the sequence homology model of TmrAB into the density map of the TmrAB–AH5 complex. TmrA is coloured in salmon, and TmrB is coloured in blue. Intracellular loop 4 is coloured in green. c, Two subunits are arranged with a pseudo-two-fold symmetry.

Extended Data Figure 8 AH5 and BA6 inhibit the ATPase activity of the TmrAB.

a, ATP standard for b–e. b–e, ATP hydrolysis assay at 37 °C. Reactions were performed at 37 °C for 20 min with 6.25 μM of TmrAB, 250 μM ATP and 2 mM MgCl2 in the presence of 25 μM of AH5 (c), BA6 (d) or a negative control Fab, U33 (e). ATP hydrolysis by TmrAB was reduced in the presence of AH5 or BA6 compared with the equivalent reaction in the absence of Fabs (b). ATP hydrolysis was not affected by the presence of U33, which does not bind to TmrAB (e). f, ATP and ADP standards (250 µM each) for g. Two peaks were resolved corresponding to ATP and ADP (black and red curves respectively). g, ATP hydrolysis by TmrAB was performed with 70 nM of TmrAB, 250 µM ATP and 2 mM MgCl2 at 60 °C for 30 min. h, Identification of the TmrAB nucleotide-binding state. ATP was not detected from the protein-extracted aqueous phase (red curve). ATP at an equivalent concentration (blue curve) is shown as a control to demonstrate sufficient sensitivity for nucleotide detection.

Extended Data Figure 9 Cysteine cross-linking validating the interaction between the C-terminal helices of TmrAB in the nucleotide-free state.

a, Three samples (marked with an asterisk in Fig. 3d) were visualized by negative-stain electron microscopy, showing that TmrAB with the double cysteine mutation has the native dimeric shape of TmrAB. TmrAB contains an exposed native cysteine residue (TmrA-C416) that could not be removed. It causes some inter-dimer cross-linking (marked by arrows) under the oxidative condition. Scale bar, 50 nm. b, Analytical HPLC demonstrating that purified TmrAB containing the A591C/A567C mutation is nucleotide free. c, ATP hydrolysis assay indicating that disulphide cross-linking inhibits the ATPase activity of TmrAB containing the double cysteine mutation. Assays were performed in triplicate for 1 h at 60 °C with 70 nM reduced or oxidized TmrAB, 250 μM ATP, and 2 mM MgCl2 before analysis by analytical HPLC.

Extended Data Table 1 Summary of TmrAB structure determination by single-particle cryo-EM

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Kim, J., Wu, S., Tomasiak, T. et al. Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter.Nature 517, 396–400 (2015). https://doi.org/10.1038/nature13872

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