Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.
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A periplasmic cinched protein is required for siderophore secretion and virulence of Mycobacterium tuberculosis
Nature Communications Open Access 26 April 2022
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The crystal structures of the SID and IrtAB-Syb_NL5 have been deposited in the Protein Data Bank (PDB) under entries 6TEK and 6TEJ, respectively The cryo-EM map has been deposited in the Electron Microscopy Data Bank under accession number EMD-10319. Plasmids, strains, the sybody Syb_NL5 and raw data are available from the authors upon reasonable request.
De Voss, J. J. et al. The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc. Natl Acad. Sci. USA 97, 1252–1257 (2000).
Snow, G. A. & White, A. J. Chemical and biological properties of mycobactins isolated from various mycobacteria. Biochem. J. 115, 1031–1045 (1969).
Gobin, J. et al. Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins. Proc. Natl Acad. Sci. USA 92, 5189–5193 (1995).
Rodriguez, G. M. & Smith, I. Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis. J. Bacteriol. 188, 424–430 (2006).
Ryndak, M. B., Wang, S., Smith, I. & Rodriguez, G. M. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain. J. Bacteriol. 192, 861–869 (2010).
Wells, R. M. et al. Discovery of a siderophore export system essential for virulence of Mycobacterium tuberculosis. PLoS Pathog. 9, e1003120 (2013).
Siegrist, M. S. et al. Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc. Natl Acad. Sci. USA 106, 18792–18797 (2009).
Tufariello, J. M. et al. Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence. Proc. Natl Acad. Sci. USA 113, E348–E357 (2016).
Locher, K. P. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat. Struct. Mol. Biol. 23, 487–493 (2016).
ter Beek, J., Guskov, A. & Slotboom, D. J. Structural diversity of ABC transporters. J. Gen. Physiol. 143, 419–435 (2014).
Zimmermann, I. et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife 7, e34317 (2018).
Egloff, P. et al. Engineered peptide barcodes for in-depth analyses of binding protein libraries. Nat. Methods 16, 421–428 (2019).
Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).
Hutter, C. A. J. et al. The extracellular gate shapes the energy profile of an ABC exporter. Nat. Commun. 10, 2260 (2019).
Hohl, M., Briand, C., Grütter, M. G. & Seeger, M. A. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nat. Struct. Mol. Biol. 19, 395–402 (2012).
Miethke, M., Hou, J. & Marahiel, M. A. The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli. Biochemistry 50, 10951–10964 (2011).
Hürlimann, L. M. et al. The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis. Antimicrob. Agents Chemother. 60, 5400–5411 (2016).
Ambudkar, S. V. et al. Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc. Natl Acad. Sci. USA 89, 8472–8476 (1992).
Lee, J. Y., Yang, J. G., Zhitnitsky, D., Lewinson, O. & Rees, D. C. Structural basis for heavy metal detoxification by an Atm1-type ABC exporter. Science 343, 1133–1136 (2014).
Tombline, G., Bartholomew, L. A., Urbatsch, I. L. & Senior, A. E. Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J. Biol. Chem. 279, 31212–31220 (2004).
Al-Shawi, M. K., Polar, M. K., Omote, H. & Figler, R. A. Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein. J. Biol. Chem. 278, 52629–52640 (2003).
Geertsma, E. R., Nik Mahmood, N. A., Schuurman-Wolters, G. K. & Poolman, B. Membrane reconstitution of ABC transporters and assays of translocator function. Nat. Protoc. 3, 256–266 (2008).
Dragset, M. S. et al. Genome-wide phenotypic profiling identifies and categorizes genes required for mycobacterial low iron fitness. Sci. Rep. 9, 11394 (2019).
Arnold, F. M. et al. A uniform cloning platform for mycobacterial genetics and protein production. Sci. Rep. 8, 9539 (2018).
Prados-Rosales, R. et al. Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition. J. Bacteriol. 196, 1250–1256 (2014).
Quazi, F., Lenevich, S. & Molday, R. S. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer. Nat. Commun. 3, 925 (2012).
Xu, D. et al. Cryo-EM structure of human lysosomal cobalamin exporter ABCD4. Cell Res. 29, 1039–1041 (2019).
Santelia, D. et al. MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development. FEBS Lett. 579, 5399–5406 (2005).
Perez, C. et al. Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature 524, 433–438 (2015).
Ratledge, C. & Ewing, M. The occurrence of carboxymycobactin, the siderophore of pathogenic mycobacteria, as a second extracellular siderophore in Mycobacterium smegmatis. Microbiology 142, 2207–2212 (1996).
Geertsma, E. R. & Dutzler, R. A versatile and efficient high-throughput cloning tool for structural biology. Biochemistry 50, 3272–3278 (2011).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Moeller, A. et al. Distinct conformational spectrum of homologous multidrug ABC transporters. Structure 23, 450–460 (2015).
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Scheres, S. H. W. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).
Heymann, J. B. & Belnap, D. M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).
Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat Methods 14, 793–796 (2017).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Voss, N. R. & Gerstein, M. 3V: cavity, channel and cleft volume calculator and extractor. Nucleic Acids Res. 38, W555–W562 (2010).
Bukowska, M. A. et al. A transporter motor taken apart: flexibility in the nucleotide binding domains of a heterodimeric ABC exporter. Biochemistry 54, 3086–3099 (2015).
Hohl, M. et al. Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG. Mol. Microbiol. 111, 1263–1282 (2019).
Hohl, M. et al. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter. Proc. Natl Acad. Sci. USA 111, 11025–11030 (2014).
We thank all members of the Seeger laboratory for discussions; B. Blattmann, C. Stutz-Ducommun and S. Eberle of the Protein Crystallization Center UZH for performing the crystallization screening; the staff of the SLS beamlines X06SA and X06DA for their support during data collection; and J. Sobeck of the Functional Genomics Center Zurich for support during SPR measurements. We also thank all members of the Medalia laboratory, especially M. Eibauer, for discussions and the Center for microscopy and image analysis (ZMB) of the UZH for providing help and equipment. Work in the laboratory of M.A.S. was supported by the European Research Council (ERC) (consolidator grant no. 772190), a SNSF Professorship of the Swiss National Science Foundation (PP00P3_144823) and a grant of the Novartis Foundation for Medical-Biological Research. Work in the laboratory of O.M. was supported by the Swiss National Science Foundation (grant 31003A_179418) and the Mäxi Foundation. Work in the laboratory of G.M. was supported by the Robert A. Welch Foundation (grant no. AT-1935-20170325) and by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM128704). F.M.A., M.S.W. and I.G. were supported by three Candoc fellowships of the University of Zurich.
The authors declare no competing interests.
Peer review information Nature thanks Gregory Cook, Damian Ekiert, Georgia Isom 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 Sequence identity of IrtAB homologues and crystal structure of IrtAB determined with the aid of a sybody.
a, Sequence identity matrix of full-length IrtAB from M. thermoresistibile (Mth), M. smegmatis (Msm) and M. tuberculosis (Mtb). b, IrtAB(ΔSID) of M. thermoresistibile (lacking the SID and the linker between the SID and the TMD of IrtA) was crystallized with the aid of a sybody. IrtA is coloured in turquoise, IrtB in purple and the sybody in grey with CDR1, CDR2 and CDR3 coloured in yellow, orange and red, respectively. CDR, complementary determining region.
Extended Data Fig. 2 Cryo-EM data processing of IrtAB.
a, Typical motion-corrected cryo-EM micrograph of the detergent-solubilized IrtAB sample. Scale bar, 50 nm. b, Representative 2D class averages used for 3D reconstruction, showing side and bottom views. c, The data-processing workflow. High-quality micrographs (2,168) were selected after motion correction and used for autopicking of 335,045 particles. Several rounds of 2D classification were performed to remove false positives, yielding 167,781 IrtAB particles, which were subjected to 3D classification. One out of six classes (indicated by the black box), containing 70,257 particles, revealed distinct structural features of IrtAB with its SID and was subjected to 3D refinement. The resulting structure was used to subtract the detergent micelle from the raw particles. Afterwards, a focused 3D refinement without the detergent micelle was performed. The resulting map was sharpened using a B-factor of −319 Å2. The final structure was resolved to 6.88 Å and reveals details of the TMDs, the NBDs and the SID.
Extended Data Fig. 3 Cryo-EM data validation.
a, Angular distribution plot of all IrtAB particles that contributed to the final map. The map and the angular distribution plot have the same orientation. The height and colour (from blue to red) of the cylinder bars is proportional to the number of particles in those views. b, Plot of the directional FSC that represents a measure of directional resolution anisotropy. Shown are the global FSC (red line), the spread of directional resolution values defined by ±1 standard deviation from the mean of the directional resolutions (area encompassed by the green dotted lines) and a histogram of 100 directional resolutions evenly sampled over the 3D FSC (blue bars). A sphericity of 0.978 was determined at an FSC threshold of 0.5, which indicates very isotropic angular distribution (a value of 1 stands for completely isotropic angular distribution). The global resolution was determined to 6.9 Å (0.143 threshold). Directional FSC determination was performed with the 3DFSC software. c, FSC plot of the final density map of IrtAB. The plot shows the unmasked (green), masked (blue), phase randomized (red) and masking-effect-corrected (black) FSC curves. The resolution at which the gold-standard FSC curve drops below the 0.143 threshold is indicated. d, Local resolution variations in the cryo-EM map. The resolution ranges from 5.5 to 10.8 Å, as calculated by BlocRes.
Extended Data Fig. 4 Superimpositions with ABC exporter structures and structural comparison of IrtAB and TM287/288.
a, Superimposition of ABC exporters using the MatchMaker tool of Chimera. Nidelmann–Wunsch alignment algorithm and Blossom-62 matrix was used. r.m.s.d. (Å2) values were calculated over the entire polypeptide chains—that is, without pruning of long atom pairs. In the case that the asymmetric unit contained several polypeptides of identical sequence, chain A was taken for analysis (indicated as ‘A’ in the protein name). IF, inward-facing; OCC, occluded; OF, outward-facing. b, Structural deviations between IrtAB and the ABC exporter TM287/288 (PDB: 4Q4H). Shown is the structure of IrtAB in an ‘open-book representation’ with the entire transporter in the middle and the two half-transporters on the left (IrtB with domain-swap-helices TMH4 and TMH5 of IrtA) and on the right (IrtA with domain-swap-helices of IrtB). The structures are shown in sausage representation in which the thickness and colour gradient from blue to red indicate variable degrees of structural deviations between IrtAB and TM287/288. Major structural deviations in the TMDs of IrtB are indicated.
Extended Data Fig. 5 Full-length IrtAB structure shown in the membrane context.
MBT molecules with a C17 aliphatic chain length are shown in stick representation. The SID is orientated towards the membrane with its mycobactin binding pocket (yellow ellipsoid) facing the inner leaflet. IrtAB is coloured as in Fig. 1.
Extended Data Fig. 6 ATPase activity measurements using HPLC-purified mycobactins of defined masses.
a, Reversed-phase chromatography separation by HPLC of Fe-cMBT (top) and Fe-MBT (bottom) isolated from M. smegmatis. Masses of mycobactins giving rise to the main peaks were determined by mass spectrometry. These analyses were performed once. b, Chemical structures of Fe-cMBT and Fe-MBT. c, ATPase activities of nanodisc-reconstituted IrtAB measured in the presence of Fe-cMBT or Fe-MBT of defined mass isolated by HPLC. Crude extracts of mycobactins were included as controls. Mycobactins were added at a concentration of 5 μM. Data points are technical triplicates, which were used to calculate the mean value (black bar). d, ATPase stimulation curves determined over a range of mycobactin concentrations for HPLC-purified samples of defined mass or the respective crude extracts. Data points are technical triplicates and curves cross through the mean values.
Extended Data Fig. 7 Mycobactin reduction by purified SID.
a, Reduction of Fe(iii)-MBT (100 μM) by M. thermoresistibile and M. smegmatis SIDs using NADPH as an electron donor and ferene as a reporter probe of released Fe(ii) (Amax = 590 nm). The 3×E mutant SID(3×E) served as a negative control. Representative data of biological duplicates are shown. b, Reduction of Fe(iii)-MBT by the SID of M. thermoresistibile performed at different concentrations of Fe(iii)-MBT. The data were fitted using the Michaelis–Menten equation. Data points are technical replicates.
Extended Data Fig. 8 Siderophore-dependent growth assay.
When grown in minimal medium (MM) under controlled iron concentrations, wild-type M. smegmatis, the M. smegmatis ΔfxbAΔmbtD double mutant (DKO) and the M. smegmatis ΔfxbAΔmbtDΔirtAB triple mutant (TKO) showed no significant differences in growth. Upon addition of the weak iron chelator 2,2′-dipyridyl, only the wild-type strain was able to grow owing to its ability to synthesize siderophores that extract iron bound to 2,2′-dipyridyl. Data points are technical triplicates and curves cross through the mean values.
Supplementary Table 1
A list of primer sequences.
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Arnold, F.M., Weber, M.S., Gonda, I. et al. The ABC exporter IrtAB imports and reduces mycobacterial siderophores. Nature 580, 413–417 (2020). https://doi.org/10.1038/s41586-020-2136-9
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