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An oligopeptide permease, OppABCD, requires an iron–sulfur cluster domain for functionality

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Oligopeptide permease, OppABCD, belongs to the type I ABC transporter family. Its role is to import oligopeptides into bacteria for nutrient uptake and to modulate the host immune response. OppABCD consists of a cluster C substrate-binding protein (SBP), OppA, membrane-spanning OppB and OppC subunits, and an ATPase, OppD, that contains two nucleotide-binding domains (NBDs). Here, using cryo-electron microscopy, we determined the high-resolution structures of Mycobacterium tuberculosis OppABCD in the resting state, oligopeptide-bound pre-translocation state, AMPPNP-bound pre-catalytic intermediate state and ATP-bound catalytic intermediate state. The structures show an assembly of a cluster C SBP with its ABC translocator and a functionally required [4Fe–4S] cluster-binding domain in OppD. Moreover, the ATP-bound OppABCD structure has an outward-occluded conformation, although no substrate was observed in the transmembrane cavity. Here, we reveal an oligopeptide recognition and translocation mechanism of OppABCD, which provides a perspective on how this and other type I ABC importers facilitate bulk substrate transfer across the lipid bilayer.

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Fig. 1: Overall architecture of substrate-bound OppABCD.
Fig. 2: Structure of OppABCD in the resting state.
Fig. 3: Structure of AMPPNP-bound OppABCD.
Fig. 4: Structure of ATP-bound OppABCD in the outward-occluded state.
Fig. 5: Nucleotide-induced conformational changes of OppABCD.
Fig. 6: Proposed oligopeptide translocation mechanism of OppABCD.

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Data availability

All data are available in the manuscript. The Electron Microscopy Data Bank (EMDB) accession codes for the 3D cryo-EM density maps reported in this paper are EMD-35990, EMD-35991, EMD-35992 and EMD-35993. Atomic coordinates and structure factors for the OppABCD, OppAW491ABCD, OppABCDE-D-AMPPNP-Mg2+, OppABCDE-D-ATP-Mg2+ and OppA53–591aa structures have been deposited in the Protein Data Bank (PDB) under accession codes 8J5Q, 8J5R, 8J5S, 8J5T and 8J5U. Source data are provided with this paper.

References

  1. Davidson Amy, L., Dassa, E., Orelle, C. & Chen, J. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. Rev. 72, 317–364 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Thomas, C. & Tampé, R. Structural and mechanistic principles of ABC transporters. Annu. Rev. Biochem. 89, 605–636 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Davidson, A. L. & Chen, J. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem. 73, 241–268 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Rice, A. J., Park, A. & Pinkett, H. W. Diversity in ABC transporters: type I, II and III importers. Crit. Rev. Biochem. Mol. Biol. 49, 426–437 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen, J. Molecular mechanism of the Escherichia coli maltose transporter. Curr. Opin. Struct. Biol. 23, 492–498 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Liu, F. et al. Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC. Sci. Adv. 6, eabb9833 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Korkhov, V. M., Mireku, S. A. & Locher, K. P. Structure of AMP-PNP-bound vitamin B12 transporter BtuCD–F. Nature 490, 367–372 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Slamti, L. & Lereclus, D. The oligopeptide ABC-importers are essential communication channels in Gram-positive bacteria. Res. Microbiol. 170, 338–344 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Monnet, V. Bacterial oligopeptide-binding proteins. Cell. Mol. Life Sci. 60, 2100–2114 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Doeven, M. K., Abele, R., Tampé, R. & Poolman, B. The binding specificity of OppA determines the selectivity of the oligopeptide ATP-binding cassette transporter. J. Biol. Chem. 279, 32301–32307 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Edwards Adrianne, N., Nawrocki Kathryn, L. & McBride Shonna, M. Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile. Infect. Immun. 82, 4276–4291 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Moraes, P. M. R. O. et al. Characterization of the Opp peptide transporter of Corynebacterium pseudotuberculosis and its role in virulence and pathogenicity. BioMed. Res. Int. 2014, 489782 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Yu, D. et al. Diversity and evolution of oligopeptide permease systems in staphylococcal species. Genomics 104, 8–13 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Dasgupta, A. et al. An oligopeptide transporter of Mycobacterium tuberculosis regulates cytokine release and apoptosis of infected macrophages. PLoS ONE 5, e12225 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Green, R. M., Seth, A. & Connell, N. D. A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione. Infect. Immun. 68, 429 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cole, S. T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Cassio Barreto de Oliveira, M. & Balan, A. The ATP-binding cassette (ABC) transport systems in Mycobacterium tuberculosis: structure, function, and possible targets for therapeutics. Biology 9, 443 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Soni, D. K., Dubey, S. K. & Bhatnagar, R. ATP-binding cassette (ABC) import systems of Mycobacterium tuberculosis: target for drug and vaccine development. Emerg. Microbes Infect. 9, 207–220 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sutcliffe, I. C. & Harrington, D. J. Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components. FEMS Microbiol. Rev. 28, 645–659 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Berntsson, R. P. A., Smits, S. H. J., Schmitt, L., Slotboom, D.-J. & Poolman, B. A structural classification of substrate-binding proteins. FEBS Lett. 584, 2606–2617 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Scheepers, G. H., Lycklama a Nijeholt, J. A. & Poolman, B. An updated structural classification of substrate-binding proteins. FEBS Lett. 590, 4393–4401 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Mitra, A., Ko, Y.-H., Cingolani, G. & Niederweis, M. Heme and hemoglobin utilization by Mycobacterium tuberculosis. Nat. Commun. 10, 4260 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Berntsson, R. P. A. et al. The structural basis for peptide selection by the transport receptor OppA. EMBO J. 28, 1332–1340 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tame, J. R. H. et al. The structural basis of sequence-independent peptide binding by OppA protein. Science 264, 1578–1581 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Tanaka, K. J. & Pinkett, H. W. Oligopeptide-binding protein from nontypeable Haemophilus influenzae has ligand-specific sites to accommodate peptides and heme in the binding pocket. J. Biol. Chem. 294, 1070–1082 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Klepsch, M. M. et al. Escherichia coli peptide binding protein OppA has a preference for positively charged peptides. J. Mol. Biol. 414, 75–85 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Mao, B., Pear, M. R., McCammon, J. A. & Quiocho, F. A. Hinge-bending in l-arabinose-binding protein. The “Venus’s-flytrap” model. J. Biol. Chem. 257, 1131–1133 (1982).

    Article  CAS  PubMed  Google Scholar 

  29. Hughes, A., Wilson, S., Dodson, E. J., Turkenburg, J. P. & Wilkinson, A. J. Crystal structure of the putative peptide-binding protein AppA from Clostridium difficile. Acta Crystallogr. F. Struct. Biol. Commun. 75, 246–253 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li, X. et al. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain. Acta Crystallogr. D Biol. Crystallogr. 69, 256–265 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Hollenstein, K., Frei, D. C. & Locher, K. P. Structure of an ABC transporter in complex with its binding protein. Nature 446, 213–216 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Sharaf, N. G. et al. Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction. eLife 10, e69742 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L. & Chen, J. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Hollenstein, K., Dawson, R. J. P. & Locher, K. P. Structure and mechanism of ABC transporter proteins. Curr. Opin. Struct. Biol. 17, 412–418 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Biemans-Oldehinkel, E., Doeven, M. K. & Poolman, B. ABC transporter architecture and regulatory roles of accessory domains. FEBS Lett. 580, 1023–1035 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Srikant, S. Evolutionary history of ATP-binding cassette proteins. FEBS Lett. 594, 3882–3897 (2020).

    Article  CAS  PubMed  Google Scholar 

  37. Smith, P. C. et al. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10, 139–149 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Procko, E., O’Mara, M. L., Bennett, W. F. D., Tieleman, D. P. & Gaudet, R. The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter. FASEB J. 23, 1287–1302 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Seyffer, F. & Tampé, R. ABC transporters in adaptive immunity. Biochim. Biophys. Acta 1850, 449–460 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. 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).

    Article  CAS  PubMed  Google Scholar 

  41. Rezwan, M., Grau, T., Tschumi, A. & Sander, P. Lipoprotein synthesis in mycobacteria. Microbiology 153, 652–658 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Singh, R., Liechti, G., Slade, J. A. & Maurelli, A. T. Chlamydia trachomatis oligopeptide transporter performs dual functions of oligopeptide transport and peptidoglycan recycling. Infect. Immun. 88, e00086-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Maio, A., Brandi, L., Donadio, S. & Gualerzi, C. O. The oligopeptide permease Opp mediates illicit transport of the bacterial P-site decoding inhibitor GE81112. Antibiotics 5, 17 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Fabbretti, A. et al. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding. Proc. Natl Acad. Sci. USA 113, E2286–E2295 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Fernando, D. M. et al. Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket. Tuberculosis 132, 102157 (2022).

    Article  CAS  PubMed  Google Scholar 

  46. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966).

    Article  CAS  PubMed  Google Scholar 

  47. Naoe, Y. et al. Crystal structure of bacterial haem importer complex in the inward-facing conformation. Nat. Commun. 7, 13411 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Nguyen, P. T., Lai, J. Y., Lee, A. T., Kaiser, J. T. & Rees, D. C. Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP binding cassette (ABC) transporter. Proc. Natl Acad. Sci. USA 115, E10596–E10604 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sikkema, H. R. et al. Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA. Sci. Adv. 6, eabd7697 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kiley, P. J. & Beinert, H. The role of Fe–S proteins in sensing and regulation in bacteria. Curr. Opin. Microbiol. 6, 181–185 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Johnson, D. C., Dean, D. R., Smith, A. D. & Johnson, M. K. Structure, function, and formation of biological iron–sulfur clusters. Annu. Rev. Biochem. 74, 247–281 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y. & Mastronarde, D. N. Software tools for automated transmission electron microscopy. Nat. Methods 16, 471–477 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. 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).

    Article  CAS  PubMed  Google Scholar 

  57. Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022).

    Article  CAS  PubMed  Google Scholar 

  58. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  62. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Stookey, L. L. Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 42, 779–781 (1970).

    Article  CAS  Google Scholar 

  66. Ter Beek, J. et al. Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ. Nucleic Acids Res. 47, 5712–5722 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the staff at the Electron Microscopy Facility of ShanghaiTech University and the beamlines BL19U1 and BL17UM at the Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data collection. We are thankful to the analytical chemistry platform at the Shanghai Institute for Advanced Immunochemical Studies (SIAIS) and the mass spectrometry system at the National Facility for Protein Science in Shanghai (NFPS) for their assistance in mass spectrometry analysis, and C. Tian and L. Yu at the high magnetic field laboratory of the Chinese Academy of Sciences for technical support and discussion. This work was supported by grants from the National Natural Science Foundation of China (grant nos. 32200996 to Xiaolin Yang, 32171217 to B.Z. and 32394010 to Z.R.), the National Key Research and Development Program of China (grant no. 2022YFC2302900 to H.Y.), the Young Elite Scientists Sponsorship Program by CAST (grant no. 2021QNRC001 to B.Z.), the Shanghai Sailing Program (grant no. 21YF1429700 to B.Z.), the Shanghai Municipal Science and Technology Major Project (grant no. ZD2021CY001 to H.Y.), the Science and Technology Commission of Shanghai Municipality (grant no. 20XD1422900 to H.Y.), the Shenzhen High-level Hospital Construction Fund (grant no. 23264G1001 to Y.Z.), the Shenzhen Clinical Research Center for Tuberculosis (grant no. 20210617141509001 to Y.Z.), the Inner Mongolia Autonomous Region Natural Science Fund Project (grant no. 2023SHZR0832 to X.Z.) and the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University.

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Author notes

  1. These authors contributed equally: Xiaolin Yang, Tianyu Hu.

Authors and Affiliations

  1. Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Xiaolin Yang, Tianyu Hu, Zhenli Lin, Yan Gao, Shan Sun, Xiuna Yang, Haitao Yang, Zihe Rao & Bing Zhang

  2. National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, China

    Xiaolin Yang & Yao Zhao

  3. State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China

    Jingxi Liang & Zihe Rao

  4. Laboratory of Structural Biology, Tsinghua University, Beijing, China

    Zhiqi Xiong & Zihe Rao

  5. The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, China

    Xiaoting Zhou

  6. Shanghai Clinical Research and Trial Center, Shanghai, China

    Xiuna Yang, Haitao Yang & Bing Zhang

  7. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane Queensland, Australia

    Luke W. Guddat

  8. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Zihe Rao

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  1. Xiaolin Yang
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Contributions

B.Z. and Z.R. conceived, initiated and coordinated the project. Xiaolin Yang purified the proteins and prepared cryo-EM samples. Xiaolin Yang and T.H. collected and processed cryo-EM data. Xiaolin Yang and B.Z. built and refined the structure model. Xiaolin Yang performed functional experiments with help from T.H., Z.L., J.L., Z.X., Y.Z., X.Z., Y.G., S.S. and Xiuna Yang. The manuscript was written by Xiaolin Yang, B.Z., L.W.G. and Z.R. with the help of H.Y. and all the other authors. All authors discussed the experiments and results and read and approved the manuscript.

Corresponding authors

Correspondence to Xiaolin Yang, Haitao Yang, Zihe Rao or Bing Zhang.

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Nature Structural & Molecular Biology thanks Jochen Zimmer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Protein purification and ATPase activity characterization.

a, Size exclusion chromatography of OppABCD, OppAW491ABCD, OppABCDE-D and OppA53-591aa samples, respectively. SDS-PAGE analysis results are also shown. b, ATPase activity of OppABCD plotted against varying ATP concentrations. c, ATPase activities of wild-type OppABCD (WT), OppAW491ABCD (APO) and OppABCDE-D (E2D). d, e, ATPase activities of OppABCD and OppAW491ABCD, respectively, in the presence of 2 mM GSH, Tri-DAP, GE81112, bradykinin, synthesized octapeptide (SLKVSVDP), synthesized nonapeptide-1 (ASPFSPDPP), nonapeptide-2 (ALPVSTDPA) and nonapeptide-3 (ALPVSVDPA). The ATPase rates in d and e are normalized based on the values of OppABCD ( ~ 200 nmol mg-1 min-1) and OppAW491ABCD (~65 nmol mg-1 min-1), respectively. In b-e, error bars represent mean ± SD based on three independent measurements. f, Heat map of ATPase activities of OppABCD and OppAW491ABCD in the presence of peptides with different lengths. Uncropped images for panel a are available as Source Data. Statistics for panels b-f are available as Source Data.

Source data

Extended Data Fig. 2 Cryo-EM data processing summary.

a-d, Flow chart of data processing for particles of OppABCD, OppAW491ABCD, OppABCDE-D-AMPPNP-Mg2+, and OppABCDE-D-ATP-Mg2+, respectively. e-h, Map-to-model FSC plot and two viewing angles of model fit to map.

Extended Data Fig. 3 Model fitting of OppABCD.

a-d, Cryo-EM density of representative regions of wild-type OppA, OppD, OppB and OppC, respectively. e, Cryo-EM map of AMPPNP-Mg2+ in OppABCDE-D-AMPPNP-Mg2+ structure. f, Cryo-EM map of ATP-Mg2+ in OppABCDE-D-ATP-Mg2+ structure. The volume threshold of the cryo-EM maps was selected at 0.385 σ.

Extended Data Fig. 4 Interfaces of OppABCD in the pre-translocation state.

a, Gate residues on TMH4/5s in OppB and OppC in the pre-translocation state. TM helices are shown as cartoons, and the sidechains of the gate residues are shown as sticks and transparent spheres. b, e, Interfaces between chains are shown as colored areas. Substrate-bound OppABCD is shown as gray surface, and contact areas of OppA, OppB, OppC and OppD are colored in pink, green, cyan and wheat, respectively. c, f, The interface between OppB (green, orange α-helical hairpin) and OppA (pink) in front and back views, respectively. d, The interface between OppC (cyan) and OppA (pink). Hydrogen bonds are indicated by yellow dashed lines.

Extended Data Fig. 5 Characterization of the Fe-S cluster of OppD.

a, Sequence alignment of N-terminal OppD from Mtb and DppD from T. tengcongensis. Conserved cysteines are marked by green stars. b, Structural alignment of OppD (yellow cartoons) and DppD (grey cartoons, PDB: 4FWI). c, UV-visible absorbance spectroscopic analysis for wild-type OppABCD. d, ATPase activities of OppABCD in the presence of L-ascorbic acid (Vc), sodium dithionite (DT) and hydrogen peroxide H2O2. Error bars represent mean ± SD based on three independent measurements. e, SDS-PAGE analysis of purified wild-type OppABCD (WT) and four variants (C286S, C292S, C299S and C317S) proteins. f, ATPase hydrolysis rates of WT and four cysteine variants. Error bars represent mean ± SD based on three independent measurements. g, ATPase hydrolysis rates (left, blue) and iron chelation analysis (right, dark yellow) of WT OppABCD and Fe-S cluster domain-deleted OppABCD(∆271-333aa) (∆FeS Domain). Error bars for the rate of ATPase hydrolysis represent the mean ± SD of three independent measurements. Error bars for iron chelation analysis of two samples represent mean ± SD based on six and four independent measurements, respectively. h, Size exclusion chromatography and SDS-PAGE analysis of OppABCD(∆271-333aa). i, Representative micrographs and 2D classes of OppABCD(∆271-333aa). Statistics for panels d, f and g are available as Source Data. Uncropped images for panels e and h are available as Source Data.

Source data

Extended Data Fig. 6 Sequence alignment of OppD homologs.

a, Sequence alignment of Mtb OppD homologs containing the Fe-S cluster domain. The four conserved cysteines in the Fe-S cluster domain are highlighted by green boxes and yellow stars. b, Phylogenetic analysis based on the sequence alignments in a. c, Taxonomy tree of homolog species selected for sequence alignment in a.

Extended Data Fig. 7 Captured oligopeptides in OppA.

a, Cartoon alignments of OppA structures. The crystal structure of OppA is in pink, while OppA from cryo-EM structures of wild-type OppABCD and AMPPNP-bound OppABCDE-D are shown in orange and green, respectively. Cα atoms of oligopeptides are shown as solid spheres. b, The Fo-Fc electron density of the oligopeptide from the crystal structure of OppA is shown as gray mesh and contoured at 2.0 σ. c, d, The cryo-EM electron density of endogenous oligopeptides from wild-type OppABCD and AMPPNP-bound OppABCDE-D, respectively. The map threshold is 0.35 σ. Possible sequence assignments for the cryo-EM density of the oligopeptide are also shown. e-g, Oligopeptide binding modes of OppA. Oligopeptide models are same as in b-d. Color scheme same as in a. Hydrogen bonds are indicated by black dashed lines.

Extended Data Fig. 8 Structure characterized Type I ABC importers in the catalytic intermediate state.

a, Cartoon and surface representations of five Type I importers in the catalytic intermediate state. OppABCD, MetQNI (PDB: 6CVL), MalEFGK2 (PDB: 3RLF), OpuA (PDB: 7AHD) and LpqY-SugABC (PDB: 7CAG) are colored by chain. b, Structural alignment of OppABCD, LpqY-SugABC and MalEFGK2. c, Zoom view of the scoop loops inserted into SBPs.

Extended Data Fig. 9 Identified endogenous oligopeptides by mass spectrometry.

a, Characterization of peptides complexed with OppABCD and OppAW491ABCD by mass spectrometry, respectively. Non-redundant peptide sequences with 99% accuracy are listed. b, Amino acid composition analysis of identified peptides. Mass spectrometry raw data for a are available as Supplementary Information.

Extended Data Fig. 10 Crystal structure comparison of OppA and its homologs.

a, Cartoon and surface representations of aligned OppA (red) and Mtb DppA (blue, PDB: 6E3D). b, c, Clipped surface view of OppA and Mtb DppA, respectively. d, Interactions between Mtb DppA and its endogenous tetrapeptide. Conserved binding residues are labeled. Hydrogen bonds are indicated by yellow dashed lines. e, Surface representation of the binding cavity (light purple) of closed OppA. The endogenous oligopeptide is depicted in pink solid spheres for the Cα atoms. f, Structural alignment of Mtb OppA (red cartoons) and L. lactis OppA (green cartoons, PDB: 3DRG). The bound oligopeptides are also indicated as solid spheres. g, The binding mode of the endogenous oligopeptide in Mtb OppA. h, The binding mode of bradykinin in L. lactis OppA. In g and h, aromatic residues in the binding pocket are labeled.

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Supplementary Table 1

Supplementary Table 1: Primers used in this study. Supplementary Table 2: De novo sequencing of the OppABCD-bound peptides. Supplementary Table 3: De novo sequencing of the OppAW491ABCD-bound peptides.

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Yang, X., Hu, T., Liang, J. et al. An oligopeptide permease, OppABCD, requires an iron–sulfur cluster domain for functionality. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01256-z

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