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PreprintCited by 20Drug Transport and Resistance MechanismsAdvanced Electron Microscopy Techniques and ApplicationsBacterial Genetics and Biotechnology

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Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

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Currie, Michael J., Davies, James S., Scalise, Mariafrancesca, Gulati, Ashutosh, Wright, Joshua D., Newton-Vesty, Michael C., Abeysekera, Gayan S., Subramanian, Ramaswamy, Wahlgren, Weixiao Y., Friemann, Rosmarie, Allison, Jane R., Mace, Peter D., Griffin, Michael D.W., Demeler, Borries, Wakatsuki, Soichi, Drew, David, Indiveri, Cesare, Dobson, Renwick C.J., North, Rachel A.·2024·eLife
DOI 10.7554/elife.92307.2PMC PMC10942642

Preprint — published as 10.7554/elife.92307.3 · Paper page

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Abstract

Abstract Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

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Research works printed but not linked: 82

  1. 2005, Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes, PLoS Comput Biol, 1, e27, 10.1371/journal.pcbi.0010027 · DOI
  2. 1998, Major facilitator superfamily, Microbiology and molecular biology reviews, 62, 1
  3. 1994, A functional superfamily of sodium/solute symporters, Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes, 1197, 133
  4. 2021, Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS), Chemical reviews, 121, 5289, 10.1021/acs.chemrev.0c00983 · DOI
  5. 2019, Secondary active transporters, 275
  6. 2021, Prokaryotic Solute/Sodium Symporters: Versatile Functions and Mechanisms of a Transporter Family, International Journal of Molecular Sciences, 22, 1880, 10.3390/ijms22041880 · DOI
  7. 2013, Unified theory of bacterial sialometabolism: how and why bacteria metabolize host sialic acids, International Scholarly Research Notices, 2013
  8. 2012, Nontypeable Haemophilus influenzae: the role of N-acetyl-5-neuraminic acid in biology, Front Cell Infect Microbiol, 2, 19
  9. 2010, The sialome—far more than the sum of its parts, Omics: a journal of integrative biology, 14, 455, 10.1089/omi.2009.0148 · DOI
  10. 2016, Host-derived sialic acids are an important nutrient source required for optimal bacterial fitness in vivo, MBio, 7, e02237
  11. 2005, Sialic acid transport in Haemophilus influenzae is essential for lipopolysaccharide sialylation and serum resistance and is dependent on a novel tripartite ATP-independent periplasmic transporter, Mol Microbiol, 58, 1173, 10.1111/j.1365-2958.2005.04901.x · DOI
  12. 2005, Novel sialic acid transporter of Haemophilus influenzae, Infection and immunity, 73, 5291, 10.1128/IAI.73.9.5291-5300.2005 · DOI
  13. 2000, Sialic acid metabolism’s dual function in Haemophilus influenzae, Mol Microbiol, 36, 1113, 10.1046/j.1365-2958.2000.01925.x · DOI
  14. 2010, Sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in Haemophilus influenzae and its effect on virulence, BMC Microbiol, 10, 48, 10.1186/1471-2180-10-48 · DOI
  15. 2022, Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth, ACS Chem Biol, 17, 1890, 10.1021/acschembio.2c00321 · DOI
  16. 2001, The tripartite ATP-independent periplasmic (TRAP) transporters of bacteria and archaea, FEMS microbiology reviews, 25, 405, 10.1111/j.1574-6976.2001.tb00584.x · DOI
  17. 2018, Tripartite ATP-independent periplasmic (TRAP) transporters and tripartite tricarboxylate transporters (TTT): from uptake to pathogenicity, Frontiers in Cellular and Infection Microbiology, 8, 33, 10.3389/fcimb.2018.00033 · DOI
  18. 2015, Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes, Biochemistry, 54, 909, 10.1021/bi501388y · DOI
  19. 2011, Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea, FEMS microbiology reviews, 35, 68, 10.1111/j.1574-6976.2010.00236.x · DOI
  20. 2012, The membrane proteins SiaQ and SiaM form an essential stoichiometric complex in the sialic acid tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM (VC1777–1779) from Vibrio cholerae, Journal of Biological Chemistry, 287, 3598, 10.1074/jbc.M111.281030 · DOI
  21. 2021, The InterPro protein families and domains database: 20 years on, Nucleic Acids Res, 49, D344, 10.1093/nar/gkaa977 · DOI · Paper page
  22. 2016, An updated structural classification of substrate-binding proteins, FEBS letters, 590, 4393, 10.1002/1873-3468.12445 · DOI · Paper page
  23. 2006, Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae, Journal of Biological Chemistry, 281, 22212, 10.1074/jbc.M603463200 · DOI
  24. 2008, Characterization of the N-acetyl-5-neuraminic acid-binding site of the extracytoplasmic solute receptor (SiaP) of nontypeable Haemophilus influenzae strain 2019, Journal of Biological Chemistry, 283, 855, 10.1074/jbc.M706603200 · DOI
  25. 2014, Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site, Acta Crystallographica Section D: Biological Crystallography, 70, 1801, 10.1107/S139900471400830X · DOI
  26. 2015, Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding, J Biol Chem, 290, 27113, 10.1074/jbc.M115.656603 · DOI
  27. 2022, Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter, Nature Communications, 13, 4471, 10.1038/s41467-022-31907-y · DOI · Paper page
  28. 2009, The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter, Proceedings of the National Academy of Sciences, 106, 1778, 10.1073/pnas.0809979106 · DOI
  29. 1997, Neuraminidase (sialidase) activity of Haemophilus parasuis, FEMS Microbiology Letters, 152, 269, 10.1111/j.1574-6968.1997.tb10438.x · DOI
  30. 1985, Transmembrane permeability channels across the outer membrane of Haemophilus influenzae type b, Journal of bacteriology, 162, 918, 10.1128/jb.162.3.918-924.1985 · DOI
  31. 1992, Outer Membrane Proteins PI and P2 of Haemophilus influenzae Type b: Structure and Identification of Surface-Exposed Epitopes, Journal of Infectious Diseases, 165, S86, 10.1093/infdis/165-Supplement_1-S86 · DOI
  32. 2009, NanC crystal structure, a model for outer-membrane channels of the acidic sugar-specific KdgM porin family, Journal of molecular biology, 394, 718, 10.1016/j.jmb.2009.09.054 · DOI
  33. 2023, Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter, Nature Communications, 14, 1120, 10.1038/s41467-023-36590-1 · DOI · Paper page
  34. 2021, 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM, Journal of Structural Biology, 213, 107702, 10.1016/j.jsb.2021.107702 · DOI
  35. 2007, Inference of Macromolecular Assemblies from Crystalline State, Journal of Molecular Biology, 372, 774, 10.1016/j.jmb.2007.05.022 · DOI
  36. 2017, The role of interfacial lipids in stabilizing membrane protein oligomers, Nature, 541, 421, 10.1038/nature20820 · DOI
  37. 2019, Structural basis for functional interactions in dimers of SLC26 transporters, Nature Communications, 10, 2032, 10.1038/s41467-019-10001-w · DOI
  38. 2016, The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism, Nature structural & molecular biology, 23, 256, 10.1038/nsmb.3166 · DOI
  39. 2012, Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter, Nature, 491, 622, 10.1038/nature11542 · DOI
  40. 2017, Structure and function of the divalent anion/Na+ symporter from Vibrio cholerae and a humanized variant, Nature communications, 8, 1
  41. 2015, Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps, Cell reports, 11, 61, 10.1016/j.celrep.2015.03.003 · DOI
  42. 2015, Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology, Nature Communications, 6, 1
  43. 2005, Structure and mechanism of the lactose permease, Comptes rendus biologies, 328, 557, 10.1016/j.crvi.2005.03.008 · DOI
  44. 2008, The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport, Science, 321, 810, 10.1126/science.1160406 · DOI
  45. 2011, The role of trimerization in the osmoregulated betaine transporter BetP, EMBO reports, 12, 804, 10.1038/embor.2011.102 · DOI
  46. 2004, Structure of a glutamate transporter homologue from Pyrococcus horikoshii, Nature, 431, 811, 10.1038/nature03018 · DOI
  47. 2015, Family resemblances: A common fold for some dimeric ion-coupled secondary transporters, J Gen Physiol, 146, 423, 10.1085/jgp.201511481 · DOI
  48. 2023, Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters, Biological Chemistry
  49. 2017, Covalently circularized nanodiscs for studying membrane proteins and viral entry, Nature methods, 14, 49, 10.1038/nmeth.4079 · DOI · Paper page
  50. 2018, An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins, Nature Communications, 9, 4253, 10.1038/s41467-018-06702-3 · DOI
  51. 2017, Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters, Nature Communications, 8, 13993, 10.1038/ncomms13993 · DOI
  52. 2022, Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2, Nature Structural & Molecular Biology, 29, 108, 10.1038/s41594-022-00738-2 · DOI
  53. 2013, The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1828, 864, 10.1016/j.bbamem.2012.09.009 · DOI
  54. 2018, Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site, Nature communications, 9, 1
  55. 2021, The high-energy transition state of the glutamate transporter homologue GltPh, The EMBO Journal, 40, e105415
  56. 2014, Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server, Nat Protoc, 9, 156, 10.1038/nprot.2013.172 · DOI
  57. 2009, Recent developments in the methods and applications of the bond valence model, Chem Rev, 109, 6858, 10.1021/cr900053k · DOI
  58. 2007, Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding, BMC Struct Biol, 7, 11, 10.1186/1472-6807-7-11 · DOI
  59. 2012, Antiparallel dimers of the small multidrug resistance protein EmrE are more stable than parallel dimers, J Biol Chem, 287, 26052, 10.1074/jbc.M112.357590 · DOI
  60. 2017, Stable membrane orientations of small dual-topology membrane proteins, Proc Natl Acad Sci U S A, 114, 7987, 10.1073/pnas.1706905114 · DOI
  61. 2016, Analytical Ultracentrifugation Data Analysis with UltraScan-III, 119, 10.1007/978-4-431-55985-6_8 · DOI
  62. 2006, Computing large sparse multivariate optimization problems with an application in biophysics
  63. 2010, A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape, Eur Biophys J, 39, 405, 10.1007/s00249-009-0413-5 · DOI
  64. 2007, Parsimonious regularization using genetic algorithms applied to the analysis of analytical ultracentrifugation experiments
  65. 2023, SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance, European Biophysics Journal
  66. 2018, The sodium sialic acid symporter from Staphylococcus aureus has altered substrate specificity, Frontiers in chemistry, 6, 233, 10.3389/fchem.2018.00233 · DOI
  67. 2017, Studying interactions of drugs with cell membrane nutrient transporters: new frontiers of proteoliposome nanotechnology, Current pharmaceutical design, 23, 3871
  68. 2017, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination, Nature methods, 14, 290, 10.1038/nmeth.4169 · DOI · Paper page
  69. 2019, Namdinator–automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps, IUCrJ, 6, 526, 10.1107/S2052252519007619 · DOI
  70. 2018, ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps, Acta Crystallogr D Struct Biol, 74, 519, 10.1107/S2059798318002425 · DOI
  71. 2010, Features and development of Coot, Acta Crystallogr D Biol Crystallogr, 66, 486, 10.1107/S0907444910007493 · DOI · Paper page
  72. 2021, ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis, Journal of Applied Crystallography, 54
  73. The PyMOL Molecular Graphics System, Version 2.1.1 Schrödinger, LLC.
  74. 2021, UCSF ChimeraX: Structure visualization for researchers, educators, and developers, Protein Sci, 30, 70, 10.1002/pro.3943 · DOI
  75. 2015, Calculations and publication-quality illustrations for analytical ultracentrifugation data, Methods in enzymology, 562, 109, 10.1016/bs.mie.2015.05.001 · DOI
  76. 2011, Sedimentation velocity to characterize surfactants and solubilized membrane proteins, Methods, 54, 56, 10.1016/j.ymeth.2010.11.003 · DOI
  77. 2008, Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca 2+–ATPase as an example, Nature protocols, 3, 1782, 10.1038/nprot.2008.177 · DOI
  78. 2015, AUC and small-angle scattering for membrane proteins, Methods in enzymology, 562, 257, 10.1016/bs.mie.2015.06.010 · DOI
  79. 2007, Analytical ultracentrifugation sedimentation velocity for the characterization of detergent-solubilized membrane proteins Ca++-ATPase and ExbB, Journal of biological physics, 33, 399, 10.1007/s10867-008-9058-3 · DOI
  80. 2019, Kalign 3: multiple sequence alignment of large data sets, Bioinformatics, 36, 1928
  81. 2009, Jalview Version 2—a multiple sequence alignment editor and analysis workbench, Bioinformatics, 25, 1189, 10.1093/bioinformatics/btp033 · DOI
  82. 2018, Automated map sharpening by maximization of detail and connectivity, Acta Crystallogr D Struct Biol, 74, 545, 10.1107/S2059798318004655 · DOI

Metadata

Authors
Currie, M. J., Davies, J. S., Scalise, M., Gulati, A., Wright, J. D., Newton-Vesty, M. C., Abeysekera, G. S., Subramanian, R., Wahlgren, W. Y., Friemann, R., Allison, J. R., Mace, P. D., Griffin, M. D. W., Demeler, B., Wakatsuki, S., Drew, D., Indiveri, C., Dobson, R. C. J., & North, R. A.
Affiliations
  • University of Canterbury (NZ)
  • Stockholm University (SE)
  • University of Calabria (IT)
  • Biological Sciences and Biomedical Engineering, Bindley Bioscience Center, Purdue University
  • University of Gothenburg (SE)
  • University of Auckland (NZ)
  • University of Otago (NZ)
  • The University of Melbourne (AU)
  • University of Lethbridge (CA)
  • Biological Sciences Division, SLAC National Accelerator Laboratory
  • Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IT)
  • The University of Sydney (AU)
Corresponding author
  • Dobson, Renwick C.J.
  • North, Rachel A.
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2024-01-24
Volume
12
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.92307.2
PMC
PMC10942642
OpenAlex
W4388099794
Cited by
20
References
1
Citations by year
2026: 5 · 2025: 4 · 2024: 10
FWCI
2.43
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • eLife (acceptedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Archive of Doctoral Theses and Digital Collections (University of Calabria) (submittedVersion)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by)
Funders
  • Australian Institute of Nuclear Science and Engineering Ltd. (Postgraduate Research Award)
  • Marsden Fund (UOC1506)
  • Ministry of Business, Innovation and Employment (UOCX1706)
  • Biomolecular Interaction Centre
Topics
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
TRAP transporter, Haemophilus influenzae, secondary active transport, homodimer structure, Na+ binding site, substrate binding site
MeSH terms
Adenosine Triphosphate, Bacterial Proteins, Haemophilus influenzae, N-Acetylneuraminic Acid, Cryoelectron Microscopy, Membrane Transport Proteins

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