Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB

Abstract

The adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg²⁺ shows a “head-to-tail” dimer of nucleo-tide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Keywords

ABC exporter-like importer / iron-loaded siderophore / IrtAB / Mycobacterium tuberculosis / ABC transporter

References

[1] Adams PD, Afonine PV, Bunkoczi G et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010;66:213–221.

[2] Alam A, Kowal J, Broude E et al. Structural insight into substrate and inhibitor discrimination by human P-glycoprotein. Science 2019;363:753–756.

[3] Arnold FM, Weber MS, Gonda I et al. The ABC exporter IrtAB imports and reduces mycobacterial siderophores. Nature 2020;580:413–417.

[4] Arosio P, Elia L, Poli M. Ferritin, cellular iron storage and regulation. IUBMB Life 2017;69:414–422.

[5] Begg SL. The role of metal ions in the virulence and viability of bacterial pathogens. Biochem Soc Trans 2019;47:77–87.

[6] ter Beek J, Guskov A, Slotboom DJ. Structural diversity of ABC transporters. J Gen Physiol 2014;143:419–435.

[7] Braibant M, Gilot P, Content J. The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis. FEMS Microbiol Rev 2000;24:449–467.

[8] Brock JH. Lactoferrin—50 years on. Biochem Cell Biol 2012;90: 245–251.

[9] Chen J. Molecular mechanism of the Escherichia coli maltose transporter. Curr Opin Struct Biol 2013;23:492–498.

[10] Chen VB, Arendall WB, 3rd et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 2010;66:12–21.

[11] Dawson RJ, Locher KP. Structure of a bacterial multidrug ABC transporter. Nature 2006;443:180–185.

[12] De Voss JJ, Rutter K, Schroeder BG et al. The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc Natl Acad Sci USA 2000;97:1252–1257.

[13] Emsley P, Lohkamp B, Scott WG et al. Features and development of coot. Acta Crystallogr D Biol Crystallogr 2010;66:486–501.

[14] Esser L, Zhou F, Pluchino KM et al. Structures of the multidrug trans-porter P-glycoprotein reveal asymmetric ATP binding and the mechanism of polyspecificity. J Biol Chem 2017;292:446–461.

[15] Gobin, J., Moore, C.H., Reeve, J.R., Jr., Wong, D.K., Gibson, B.W., and Horwitz, M.A. (1995). Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins. Proc Natl Acad Sci U S A 92, 5189–93.

[16] Goddard TD, Huang CC, Meng EC et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci 2018;27:14–25.

[17] Grigorieff N. Frealign: an exploratory tool for single-particle Cryo-EM. Methods Enzymol 2016;579:191–226.

[18] Hofmann S, Januliene D, Mehdipour AR et al. Conformation space of a heterodimeric ABC exporter under turnover conditions. Nature 2019;571:580–583.

[19] Hohl M, Briand C, Grutter MG et al. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nat Struct Mol Biol 2012;19:395–402.

[20] Hu W, Parkinson C, Zheng H. Mechanistic insights revealed by YbtPQ in the occluded state. bioRxiv 2021.

[21] Jin MS, Oldham ML, Zhang Q et al. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans. Nature 2012;490:566–569.

[22] Johnson ZL, Chen J. Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 2017;168:1075–1085.e9.

[23] de Jong G, van Dijk JP, van Eijk HG. The biology of transferrin. Clin Chim Acta 1990;190:1–46.

[24] Khare D, Oldham ML, Orelle C et al. Alternating access in maltose transporter mediated by rigid-body rotations. Mol Cell 2009;33:528–536.

[25] Kim Y, Chen J. Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation. Science 2018;359:915–919.

[26] Kodan A, Yamaguchi T, Nakatsu T et al. Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog. Proc Natl Acad Sci USA 2014;111:4049–4054.

[27] Koh EI, Hung CS, Parker KS et al. Metal selectivity by the virulence-associated yersiniabactin metallophore system. Metallomics 2015;7:1011–1022.

[28] Liu F, Liang J, Zhang B et al. Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC. Sci Adv 2020;6.

[29] Locher KP. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat Struct Mol Biol 2016;23:487–493.

[30] Mi W, Li Y, Yoon SH et al. Structural basis of MsbA-mediated lipopolysaccharide transport. Nature 2017;549:233–237.

[31] Oldham ML, Khare D, Quiocho FA et al. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 2007;450:515–521.

[32] Paoli M, Marles-Wright J, Smith A. Structure-function relationships in heme-proteins. DNA Cell Biol 2002;21:271–280.

[33] Perez C, Gerber S, Boilevin J et al. Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature 2015;524:433–438.

[34] Pettersen EF, Goddard TD, Huang CC et al. UCSF Chimera — a visualization system for exploratory research and analysis. J Comput Chem 2004;25:1605–1612.

[35] Procko E, O’Mara ML, Bennett WF et al. The mechanism of ABC trans-porters: general lessons from structural and functional studies of an antigenic peptide transporter. FASEB J 2009;23:1287–1302.

[36] Punjani A, Rubinstein JL, Fleet DJ et al. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 2017;14:290–296.

[37] Rempel S, Gati C, Nijland M et al. A mycobacterial ABC trans-porter mediates the uptake of hydrophilic compounds. Nature 2020;580:409–412.

[38] Rice AJ, Park A, Pinkett HW. Diversity in ABC transporters: type I, II and III importers. Crit Rev Biochem Mol Biol 2014;49:426–437.

[39] Rodriguez GM, Smith I. Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis. J Bacteriol 2006;188:424–430.

[40] Ryndak MB, Wang S, Smith I et al. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain. J Bacteriol 2010;192:861–869.

[41] Skaar EP. The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog 2010;6:e1000949.

[42] Snow GA, White AJ. Chemical and biological properties of mycobactins isolated from various mycobacteria. Biochem J 1969;115:1031–1050.

[43] Srinivasan V, Pierik AJ, Lill R. Crystal structures of nucleotide-free and glutathione-bound mitochondrial ABC transporter Atm1. Science 2014;343:1137–1140.

[44] Thomas C, Aller SG, Beis K et al. Structural and functional diversity calls for a new classification of ABC transporters. FEBS Lett 2020;594:3767–3775.

[45] Wang Z, Hu W, Zheng H. Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter. Sci Adv 2020;6:eaay7997.

[46] Wells RM, Jones CM, Xi Z et al. Discovery of a siderophore export system essential for virulence of Mycobacterium tuberculosis. PLoS Pathog 2013;9:e1003120.

[47] Xu D, Feng Z, Hou WT et al. Cryo-EM structure of human lysosomal cobalamin exporter ABCD4. Cell Res 2019;29:1039–1041.

[48] Yang R, Cui L, Hou YX et al. ATP binding to the first nucleotide binding domain of multidrug resistance-associated protein plays a regulatory role at low nucleotide concentration, whereas ATP hydrolysis at the second plays a dominant role in ATP-dependent leukotriene C4 transport. J Biol Chem 2003;278:30764–30771.

[49] Zhang, Z., and Chen, J. (2016). Atomic structure of the cystic fibrosis transmembrane conductance regulator. Cell 167, 1586–1597.e9 e1589.

[50] Zheng SQ, Palovcak E, Armache JP et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 2017;14:331–332.

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2022 The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.