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A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds

Nature volume 580, pages 409–412 (2020)Cite this article

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Abstract

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1,2,3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4,5,6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8,9,10,11.

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Fig. 1: Import of cobalamin and bleomycin by Rv1819c and ATPase assay.
Fig. 2: Structure of Mg–AMP-PNP-bound Rv1819c(E576G).

Data availability

Atomic coordinates and the cryo-EM map are deposited in the Protein Data Bank under accession numbers 6TQF (AMP-PNP) and 6TQE (purified without addition) and in the Electron Microscopy Data Bank under accession numbers EMD-10550 (AMP-PNP), EMD-10549 (purified without addition), respectively. Source Data for Fig. 1 and Extended Data Figs. 1, 6 are available with this paper. The other data from this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. Heinemann and S. Bonsing-Vedelaar for providing the strains from the Keio collection; L. Hielkema for help with purification optimization; A. Garaeva for support with screening-grid preparation and preparing lipid solutions; I. van ‘t Land-Kuper for support with growth assays and protein purifications; R. Singh for design of the codon-optimized construct; the SLAC-Stanford Cryo-EM center (SLAC National Accelerator Laboratory), where the dataset was recorded, for microscope time; E. A. Montabana for microscope support; and Y.-T. Li for support with computing resources. We acknowledge support from the cryo-EM and mass spectrometry facilities of the University of Groningen. This study was supported by the European Molecular Microbiology Organization (EMBO short-term fellowship ASTF-382-2015 to S.R.), the Netherlands Organisation for Scientific Research (NWO-TOP grant 714.018.003 to D.J.S. and NWO Vidi grant 723.014.002 to A.G.), the Swedish Research Council and the Carl Tryggers Foundation (2015-05288 and CTS17:114, respectively, to J.W.d.G), and the Department of Energy, Laboratory Directed Research and Development program at SLAC National Accelerator Laboratory, under contract DE-AC02-76SF00515.

Author information

Author notes

  1. S. Rempel

    Present address: Structural Biology and Biophysics, Biozentrum–University of Basel, Basel, Switzerland

Authors and Affiliations

  1. Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands

    S. Rempel, M. Nijland, C. Thangaratnarajah, A. Guskov & D. J. Slotboom

  2. SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    C. Gati

  3. Department of Structural Biology, School of Medicine, Stanford University, Palo Alto, CA, USA

    C. Gati

  4. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    A. Karyolaimos & J. W. de Gier

  5. Moscow Institute of Physics and Technology, Dolgoprudny, Russia

    A. Guskov

  6. Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

    D. J. Slotboom

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Contributions

All authors designed experiments. S.R. conducted cloning and growth assays, S.R., C.T. and M.N. performed biochemical assays. S.R. and A.G. built the model. S.R., A.K. and J.W.d.G. constructed knockout strains. C.G. collected and processed cryo-EM data. All authors analysed data. S.R. and D.J.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to C. Gati, A. Guskov or D. J. Slotboom.

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The authors declare no competing interests.

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Peer review information Nature thanks Damian Ekiert, Eric Rubin and Jochen Zimmer for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Biotin-dependent-growth assay with E. coli ΔbioH ΔyigM ΔsbmA::KmR and cobalamin-dependent-growth assay with additional controls.

a–d, The mutant strain expressing either Rv1819c (blue) or carrying the empty expression vector pBAD24 (yellow) was grown in the presence of 0.001 μM biotin (a), 0.01 μM biotin (b), 0.1 μM biotin (c) or 1 μM biotin (d). There is no difference between Rv1819c-expressing cells and cells without Rv1819c, whereas cells expressing the E. coli biotin transporter YigM can grow under these conditions21. The grey area shows the s.e.m. of biological triplicates. e, Growth curves for Rv1819c (blue), Rv1819c(E576G) (red) and the empty vector (pBAD24, green) are shown as in Fig. 1b. Additional control curves from the cobalamin transporter BtuM (yellow) and ABC transporter BtuCDF (orange) are included for better comparison. The grey area shows the s.e.m. of the mean (lines) of n = 3 biological triplicates.

Source data

Extended Data Fig. 2 Resolution and angular distribution of the cryo-EM 3D reconstructions.

a, Comparison of the structures obtained in the presence (left) or absence (right) of added Mg–AMP-PNP and their corresponding densities (blue mesh). Representation of nucleotides as in Fig. 2a. b, Fourier shell correlation plot for Rv1819c(E576G) Mg–AMP-PNP (314,691 particles, left) and Rv1819c(E576G) without addition of substrates (35,890 particles, right). c, Maps (without micelle belt) of both structures in front view (left) and side view (right, rotated by 90°), coloured according to local resolution, from blue (3.25 Å and 4.008 Å) to red (4.15 Å and 5.081 Å). d, Angular distribution of views in the 3D reconstruction with applied C2 symmetry (left view and right view, rotated by 180°).

Extended Data Fig. 3 Cryo-EM data-processing workflow for Rv1819c(E576G) with Mg–AMP-PNP.

a, Single-particle cryo-EM data-processing scheme using RELION3.0. b, Representative micrograph showing picked particles (indicated by green circles). c, Two-dimensional class averages show distinct secondary structure features.

Extended Data Fig. 4 Seventeen-amino-acid loop insert in TMH3, surface potential of Rv1819c and nucleotide binding domain of Rv1819c(E576G).

a, Density of Rv1819c(E576G) showing only TMH3 from both protomers (grey and pink) for clarity. The helix is interrupted by a 17-residue loop. For comparison, the density map is layered with the central cross-section of the cavity (red). b, Surface representation of Rv1819c(E576G) coloured according to electrostatic potential, showing that the extracellular cap is positively charged. c, Closed NBD homodimer (grey, chain A; pink, chain B) with one modelled Mg–AMP-PNP molecule (grey sphere, Mg2+; wheat, carbon atoms; red, oxygen atoms; blue, nitrogen atoms; orange, phosphorus atoms) in each active site. d, Protein–nucleotide interaction between the Walker A (chain B) residues Lys466 and Thr469 with AMP-PNP (density shown by blue mesh; σ = 6) and Thr467 with the Mg2+ ion. Residues Ser552 and Glu555 from the signature motif (chain A), which also bind the AMP-PNP molecule, are indicated. The E576G substitution in the Walker B motif is indicated. e, f, As c and d, for Rv1819c(E576G), which was purified without the addition of nucleotides. The observed density (blue mesh, σ = 6) in the pocket is large enough to fit a Mg–ATP molecule.

Extended Data Fig. 5 Modelled DDM molecules and density patches of unclear origin.

a–d, Densities (blue mesh) rendered at σ = 3.5 are shown, which are well-defined but cannot be assigned at the current resolution, and others that might originate from DDM molecules (wheat with oxygen atoms red). a, Two densities located near Trp122. b, Density patch at the interface of the protomers close to His159 and Gln163. c, Two densities close to Trp233. d, Five putative DDM molecules (only one protomer for clarity).

Extended Data Fig. 6 Elution profile of Rv1819c in the presence of nucleotides and cobalamin.

The wild-type protein was purified in the presence of 2 mM cobalamin and 5 mM Mg–ATP during solubilization and affinity chromatography. Elution from the affinity column was performed in the presence of 5 mM Mg–AMP-PNP. The size-exclusion chromatography buffer did not contain substrates. There was no co-elution of cobalamin, which absorbs strongly at 361 nm (red) and eluted between 18 ml and 24 ml, after the protein (280 nm, blue). The wild-type protein eluted as non-native dimers of homodimers (~10 ml elution volume) and the native homodimeric species (~12 ml elution volume).

Source data

Extended Data Fig. 7 Cobalamin and bleomycin docked into Rv1819c(E576G) and density patches inside the occluded cavity of Rv1819c(E576G).

a, b, View through chain B (pink, residues 371–393 were omitted for clarity) into the cavity of Rv1819c. Chain A (grey) is shown as surface and cartoon representation, and cobalamin and bleomycin are shown in stick representation (wheat with oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and cobalt in light pink). a, b, Docked cobalamin (a, right) and docked bleomycin (b, right). c, d, The two density patches in the cavity, rendered at σ = 5 and shown as blue mesh. For clarity, only one chain is shown. c, View of the entire chain. d, Closer view of the densities resembling a potential co-purified peptide.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 2 Primers used in the study
Full size table

Supplementary information

Supplementary Figure 1

This file contains the uncropped western blots.

Reporting Summary

41586_2020_2072_MOESM3_ESM.xlsx

Supplementary Table 1De novo sequencing results of peptides identified by mass spectrometry that may occupy the cavity of Rv1819c in our structures.

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Rempel, S., Gati, C., Nijland, M. et al. A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds. Nature 580, 409–412 (2020). https://doi.org/10.1038/s41586-020-2072-8

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