Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

  • Elena N. Kitova
  • Rambod Daneshfar
  • Paola Marcato
  • George L. Mulvey
  • Glen Armstrong
  • John S. Klassen
Article

Abstract

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B5 homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 °C and subunit concentrations ranging from 5 to 85 µM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 µM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B5 ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 °C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B5 ions at the +12 and +13 charge states were compared with values reported for the corresponding B5 ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (Ea) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the Ea values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.

References

  1. 1.
    Karmali, M. A. Infection by Shiga Toxin-Producing Escherichia coli: An Overview. Mol. Biotechnol. 2004, 26, 117–122.CrossRefGoogle Scholar
  2. 2.
    Nataro, J. P.; Kaper, J. B. Diarrheagenic. Escherichia coli. Clin. Microbiol. Rev. 1998, 11, 142–201.Google Scholar
  3. 3.
    Fraser, M. E.; Chernaia, M. M.; Kozlov, Y. V.; James, M. N. Crystal Structure of the Holotoxin From Shigella dysenteriae at 2.5 Å Resolution. Nat. Struct. Biol. 1994, 1, 59–64.CrossRefGoogle Scholar
  4. 4.
    Fraser, M. E.; Fujinaga, M.; Cherney, M. M.; Melton-Celsa, A. R.; Twiddy, E. M.; O’Brien, A. D.; James, M. N. Structure of Shiga Toxin Type 2 (Stx2) From Escherichia coli O157:H7. J. Biol. Chem. 2004, 279, 27511–27517.CrossRefGoogle Scholar
  5. 5.
    Ling, H.; Boodhoo, A.; Hazes, B.; Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J. Structure of the Shiga-like Toxin I B-Pentamer Complexed With an Analogue of its Receptor Gb3. Biochemistry 1998, 37, 1777–1788.CrossRefGoogle Scholar
  6. 6.
    Calderwood, S. B.; Auclair, F.; Donohue-Rolfe, A.; Keusch, G. T.; Mekalanos, J. J. Nucleotide Sequence of the Shiga-like Toxin Genes of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 4364–4368.CrossRefGoogle Scholar
  7. 7.
    Strockbine, N. A.; Jackson, M. P.; Sung, L. M.; Holmes, R. K.; O’Brien, A. D. Cloning and Sequencing of the Genes for Shiga Toxin From Shigella dysenteriae Type 1. J. Bacteriol. 1988, 170, 1116–1122.Google Scholar
  8. 8.
    Boodhoo, A.; Read, R. J.; Brunton, J. Crystallization and Preliminary X-Ray Crystallographic Analysis of Verotoxin-1 B-Subunit. J. Mol. Biol. 1991, 221, 729–731.CrossRefGoogle Scholar
  9. 9.
    Richardson, J. M.; Evans, P. D.; Homans, S. W.; Donohue-Rolfe, A. Solution Structure of the Carbohydrate-Binding B-Subunit Homopentamer of Verotoxin VT-1 From E. coli. Nat. Struct. Biol. 1997, 4, 190–193.CrossRefGoogle Scholar
  10. 10.
    Hashimoto, H.; Mizukoshi, K.; Nishi, M.; Kawakita, T.; Hasui, S.; Kato, Y.; Ueno, Y.; Takeya, R.; Okuda, N.; Takeda, T. Epidemic of Gastrointestinal Tract Infection Including Hemorrhagic Colitis Attributable to Shiga Toxin 1-Producing Escherichia coli O118:H2 at a Junior High School in Japan. Pediatrics 1999, 103, E2.Google Scholar
  11. 11.
    Head, S. C.; Petric, M.; Richardson, S.; Roscoe, M.; Karmali, M. A. Purification and Characterization of Verocytotoxin-2. Fems Microbiol. Lett. 1988, 51, 211–215.CrossRefGoogle Scholar
  12. 12.
    Kleanthous, H.; Smith, H. R.; Scotland, S. M.; Gross, R. J.; Rowe, B.; Taylor, C. M.; Milford, D. V. Haemolytic Uraemic Syndromes in the British Isles, 1985-8: Association With Verocytotoxin Producing Escherichia coli. Part 2: Microbiological Aspects. Arch. Dis. Child 1990, 65, 722–727.CrossRefGoogle Scholar
  13. 13.
    Ostroff, S. M.; Tarr, P. I.; Neill, M. A.; Lewis, J. H.; Hargrett-Bean, N.; Kobayashi, J. M. Toxin Genotypes and Plasmid Profiles as Determinants of Systemic Sequelae in Escherichia coli O157:H7 Infections. J. Infect. Dis. 1989, 160, 994–998.CrossRefGoogle Scholar
  14. 14.
    Scotland, S. M.; Willshaw, G. A.; Smith, H. R.; Rowe, B. Properties of Strains of Escherichia coli Belonging to Serogroup O157 With Special Reference to Production of Vero Cytotoxins VT1 and VT2. Epidemiol. Infect. 1987, 99, 613–624.CrossRefGoogle Scholar
  15. 15.
    Siegler, R. L.; Obrig, T. G.; Pysher, T. J.; Tesh, V. L.; Denkers, N. D.; Taylor, F. B. Response to Shiga Toxin 1 and 2 in a Baboon Model of Hemolytic Uremic Syndrome. Pediatr. Nephrol. 2003, 18, 92–96.Google Scholar
  16. 16.
    Tesh, V. L.; Burris, J. A.; Owens, J. W.; Gordon, V. M.; Wadolkowski, E. A.; O’Brien, A. D.; Samuel, J. E. Comparison of the Relative Toxicities of Shiga-Like Toxins Type I and Type II for Mice. Infect. Immunol. 1993, 61, 3392–3402.Google Scholar
  17. 17.
    Wadolkowski, E. A.; Sung, L. M.; Burris, J. A.; Samuel, J. E.; O’Brien, A. D. Acute Renal Tubular Necrosis and Death of Mice Orally Infected With Escherichia coli Strains That Produce Shiga-Like Toxin Type II. Infect. Immunol. 1990, 58, 3959–3965.Google Scholar
  18. 18.
    Head, S. C.; Karmali, M. A.; Lingwood, C. A. Preparation of VT1 and VT2 Hybrid Toxins From Their Purified Dissociated Subunits. Evidence for B Subunit Modulation of a Subunit Function. J. Biol. Chem. 1991, 266, 3617–3621.Google Scholar
  19. 19.
    Kitova, E. N.; Kitov, P. I.; Bundle, D. R.; Klassen, J. S. The Observation of Multivalent Complexes of Shiga-Like Toxin With Globotriaoside and the Determination of Their Stoichiometry by Nanoelectrospray Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry. Glycobiology 2001, 11, 605–611.CrossRefGoogle Scholar
  20. 20.
    Dunbar, R. C.; McMahon, T. B. Activation of Unimolecular Reactions by Ambient Blackbody Radiation. Science 1998, 279, 194–197.CrossRefGoogle Scholar
  21. 21.
    Price, W. D.; Schnier, P. D.; Jockusch, R. A.; Strittmatter, E. F.; Williams, E. R. Unimolecular Reaction Kinetics in the High-Pressure Limit Without Collisions. J. Am. Chem. Soc. 1996, 118, 10640–10644.CrossRefGoogle Scholar
  22. 22.
    Felitsyn, N. F.; Kitova, E. N.; Klassen, J. S. Thermal Decomposition of a Gaseous Multiprotein Complex Studied by Blackbody Infrared Radiative Dissociation. Investigating the Origin of the Asymmetric Dissociation Behaviour. Anal. Chem. 2001, 73, 4647–4661.CrossRefGoogle Scholar
  23. 23.
    Marcato, P.; Mulvey, G.; Read, R. J.; Vander, H. K.; Nation, P. N.; Armstrong, G. D. Immunoprophylactic Potential of Cloned Shiga Toxin 2 B Subunit. J. Infect. Dis. 2001, 183, 435–443.CrossRefGoogle Scholar
  24. 24.
    Mulvey, G.; Vanmaele, R.; Mrazek, M.; Cahill, M.; Armstrong, G. D. Affinity Purification of Shiga-Like Toxin I and Shiga-Like Toxin II. J. Microbiol. Methods 1998, 32, 247–252.CrossRefGoogle Scholar
  25. 25.
    Daneshfar, R.; Kitova, E. N.; Klassen, J. S. Determination of Protein-Ligand Association Thermochemistry Using Variable-Temperature Nanoelectrospray Mass Spectrometry. J. Am. Chem. Soc. 2004, 126, 4786–4787.CrossRefGoogle Scholar
  26. 26.
    Versluis, C.; van der Staaij, A.; Stokvis, E.; Heck, A. J. R.; de Craene, B. Metastable Ion Formation and Disparate Charge Separation in the Gas-Phase Dissection of Protein Assemblies Studied by Orthogonal Time-of-Flight Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 329–336.CrossRefGoogle Scholar
  27. 27.
    Jurchen, J. C.; Williams, E. R. Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein Homodimers. J. Am. Chem. Soc. 2003, 125, 2817–2826.CrossRefGoogle Scholar
  28. 28.
    Wang, W. J.; Kitova, E. N.; Klassen, J. S. Influence of Solution and Gas Phase Processes on Protein-Carbohydrate Binding Affinities Determined by Nanoelectrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 2003, 75, 4945–4955.CrossRefGoogle Scholar
  29. 29.
    Wang, W. J.; Kitova, E. N.; Klassen, J. S. Nonspecific Protein-Carbohydrate Complexes Produced by Nanoelectrospray Ionization. Factors Influencing Their Formation and Stability. Anal. Chem. 2005, 77, 3060–3071.CrossRefGoogle Scholar
  30. 30.
    Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.CrossRefGoogle Scholar
  31. 31.
    Nettleton, E. J.; Sunde, M.; Lai, Z. H.; Kelly, J. W.; Dobson, C. M.; Robinson, C. V. Protein Subunit Interactions and Structural Integrity of Amyloidogenic Transthyretins: Evidence From Electrospray Mass Spectrometry. J. Mol. Biol. 1998, 281, 553–564.CrossRefGoogle Scholar
  32. 32.
    Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Detection and Selective Dissociation of Intact Ribosomes in a Mass Spectrometer. Proc. Nat. Acad. Sci. U. S. A. 2000, 97, 5185–5190.CrossRefGoogle Scholar
  33. 33.
    Hanson, C. L.; Fucini, P.; Ilag, L. L.; Nierhaus, K. H.; Robinson, C. V. Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer. J. Biol. Chem. 2003, 278, 1259–1267.CrossRefGoogle Scholar
  34. 34.
    Aquilina, J. A.; Robinson, C. V. Investigating Interactions of the Pentraxins Serum Amyloid P Component and C-Reactive Protein by Mass Spectrometry. Biochem. J. 2003, 375, 323–328.CrossRefGoogle Scholar
  35. 35.
    Sobott, F.; McCammon, M. G.; Hernandez, H.; Robinson, C. V. The Flight of Macromolecular Complexes in a Mass Spectrometer. Phil. Trans. R. Soc. B 2005, 363, 379–389.CrossRefGoogle Scholar
  36. 36.
    Peschke, M.; Verkerk, U. H.; Kebarle, P. Features of the ESI Mechanism that Affect the Observation of Multiply Charged Noncovalent Protein Complexes and the Determination of the Association Constant by the Titration Method. J. Am. Soc. Mass Spectrom. 2004, 15, 1424–1434.CrossRefGoogle Scholar
  37. 37.
    Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. Observation of the Noncovalent Quaternary Associations of Proteins by Electrospray-Ionization Mass Spectrometry. J. Am. Chem. Soc. 1994, 116, 5271–5278.CrossRefGoogle Scholar
  38. 38.
    Heck, A. J. R.; van den Heuvel, R. H. H. Investigation of Intact Protein Complexes by Mass Spectrometry. Mass Spectrom. Rev. 2004, 23, 368–389.CrossRefGoogle Scholar
  39. 39.
    Fandrich, M.; Tito, M. A.; Leroux, M. R.; Rostom, A. A.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Observation of the Noncovalent Assembly and Disassembly Pathways of the Chaperone Complex MtGimC by Mass Spectrometry. Proc. Nat. Acad. Sci. U. S. A. 2000, 97, 14151–14155.CrossRefGoogle Scholar
  40. 40.
    Benesch, J. L. P.; Sobott, F.; Robinson, C. V. Thermal Dissociation of Multimeric Protein Complexes by Using Nanoelectrospray Mass Spectrometry. Anal. Chem. 2003, 75, 2208–2214.CrossRefGoogle Scholar
  41. 41.
    Lentze, N.; Aquilina, J. A.; Lindbauer, M.; Robinson, C. V.; Narberhaus, F. Temperature and Concentration-Controlled Dynamics of Rhizobial Small Heat Shock Proteins. Eur. J. Biochem. 2004, 271, 2494–2503.CrossRefGoogle Scholar
  42. 42.
    St. Hilaire, P. M.; Boyd, M. K.; Toone, E. J. Interaction of the Shiga-Like Toxin Type-1 B-Subunit With Its Carbohydrate Receptor. Biochemistry 1994, 33, 14452–14463.CrossRefGoogle Scholar
  43. 43.
    Sinha, N.; Smith-Gill, S. J. Electrostatics in Protein Binding and Function. Curr. Protein Pept. Sci. 2002, 3, 601–614.CrossRefGoogle Scholar
  44. 44.
    Yu, Y.; Monera, O. D.; Hodges, R. S.; Privalov, P. L. Ion Pairs Significantly Stabilize Coiled-Coils in the Absence of Electrolyte. J. Mol. Biol. 1996, 255, 367–372.CrossRefGoogle Scholar
  45. 45.
    Natarajan, R.; Linstedt, A. D. A Cycling cis-Golgi Protein Mediates Endosome-to-Golgi Traffic. Mol. Biol. Cell 2004, 15, 4798–4806.CrossRefGoogle Scholar
  46. 46.
    Kitova, E. N.; Bundle, D. R.; Klassen, J. S. Partitioning of Solvent Effects and Intrinsic Interactions in Biological Recognition. Angev. Chem. Int. Ed. 2004, 43, 4183–4186.CrossRefGoogle Scholar
  47. 47.
    Wang, W.; Kitova, E. N.; Klassen, J. S. Structure and Stability of Protein-Carbohydrate Complexes in the Gas Phase. Origin of Nonspecific Binding. J. Am. Soc. Mass Spectrom. 2005, 16, 1583–1594.CrossRefGoogle Scholar
  48. 48.
    Kitova, E. N.; Bundle, D. R.; Klassen, J. S. Thermal Dissociation of Protein-Oligosaccharide Complexes in the Gas Phase. Mapping the Intrinsic Intermolecular Interactions. J. Am. Chem. Soc. 2002, 124, 5902–5913.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2005

Authors and Affiliations

  • Elena N. Kitova
    • 1
  • Rambod Daneshfar
    • 1
  • Paola Marcato
    • 2
  • George L. Mulvey
    • 3
  • Glen Armstrong
    • 3
  • John S. Klassen
    • 4
  1. 1.Department of ChemistryUniversity of AlbertaEdmontonCanada
  2. 2.Department of Microbiology and ImmunologyDalhousie UniversityHalifaxCanada
  3. 3.Department of Microbiology and Infectious Diseases, Faculty of MedicineUniversity of CalgaryAlbertaCanada
  4. 4.Department of ChemistryUniversity of AlbertaEdmontonCanada

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