Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Sep;3(9):1444–1463. doi: 10.1002/pro.5560030911

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

M J Bennett 1, S Choe 1, D Eisenberg 1
PMCID: PMC2142933  PMID: 7833807

Abstract

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.

Full Text

The Full Text of this article is available as a PDF (2.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. J., Ford G. C., Koekoek R., Lentz P. J., McPherson A., Jr, Rossmann M. G., Smiley I. E., Schevitz R. W., Wonacott A. J. Structure of lactate dehydrogenase at 2-8 A resolution. Nature. 1970 Sep 12;227(5263):1098–1103. doi: 10.1038/2271098a0. [DOI] [PubMed] [Google Scholar]
  2. Baker E. N., Hubbard R. E. Hydrogen bonding in globular proteins. Prog Biophys Mol Biol. 1984;44(2):97–179. doi: 10.1016/0079-6107(84)90007-5. [DOI] [PubMed] [Google Scholar]
  3. Bennett M. J., Choe S., Eisenberg D. Domain swapping: entangling alliances between proteins. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3127–3131. doi: 10.1073/pnas.91.8.3127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blewitt M. G., Chung L. A., London E. Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration. Biochemistry. 1985 Sep 24;24(20):5458–5464. doi: 10.1021/bi00341a027. [DOI] [PubMed] [Google Scholar]
  5. Bowie J. U., Lüthy R., Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991 Jul 12;253(5016):164–170. doi: 10.1126/science.1853201. [DOI] [PubMed] [Google Scholar]
  6. Brandhuber B. J., Allured V. S., Falbel T. G., McKay D. B. Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A. Proteins. 1988;3(3):146–154. doi: 10.1002/prot.340030303. [DOI] [PubMed] [Google Scholar]
  7. Brünger A. T., Krukowski A., Erickson J. W. Slow-cooling protocols for crystallographic refinement by simulated annealing. Acta Crystallogr A. 1990 Jul 1;46(Pt 7):585–593. doi: 10.1107/s0108767390002355. [DOI] [PubMed] [Google Scholar]
  8. Carroll S. F., Barbieri J. T., Collier R. J. Dimeric form of diphtheria toxin: purification and characterization. Biochemistry. 1986 May 6;25(9):2425–2430. doi: 10.1021/bi00357a019. [DOI] [PubMed] [Google Scholar]
  9. Carroll S. F., McCloskey J. A., Crain P. F., Oppenheimer N. J., Marschner T. M., Collier R. J. Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7237–7241. doi: 10.1073/pnas.82.21.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choe S., Bennett M. J., Fujii G., Curmi P. M., Kantardjieff K. A., Collier R. J., Eisenberg D. The crystal structure of diphtheria toxin. Nature. 1992 May 21;357(6375):216–222. doi: 10.1038/357216a0. [DOI] [PubMed] [Google Scholar]
  11. Collier R. J. Diphtheria toxin: mode of action and structure. Bacteriol Rev. 1975 Mar;39(1):54–85. doi: 10.1128/br.39.1.54-85.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Collins C. M., Collier R. J. Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives. Biochim Biophys Acta. 1985 Apr 5;828(2):138–143. doi: 10.1016/0167-4838(85)90049-4. [DOI] [PubMed] [Google Scholar]
  13. Domenighini M., Montecucco C., Ripka W. C., Rappuoli R. Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding. Mol Microbiol. 1991 Jan;5(1):23–31. doi: 10.1111/j.1365-2958.1991.tb01822.x. [DOI] [PubMed] [Google Scholar]
  14. Donovan J. J., Simon M. I., Draper R. K., Montal M. Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc Natl Acad Sci U S A. 1981 Jan;78(1):172–176. doi: 10.1073/pnas.78.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Drazin R., Kandel J., Collier R. J. Structure and activity of diphtheria toxin. II. Attack by trypsin at a specific site within the intact toxin molecule. J Biol Chem. 1971 Mar 10;246(5):1504–1510. [PubMed] [Google Scholar]
  16. Dumont M. E., Richards F. M. The pH-dependent conformational change of diphtheria toxin. J Biol Chem. 1988 Feb 5;263(4):2087–2097. [PubMed] [Google Scholar]
  17. Eisenberg D., McLachlan A. D. Solvation energy in protein folding and binding. Nature. 1986 Jan 16;319(6050):199–203. doi: 10.1038/319199a0. [DOI] [PubMed] [Google Scholar]
  18. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  19. Erickson H. P. Co-operativity in protein-protein association. The structure and stability of the actin filament. J Mol Biol. 1989 Apr 5;206(3):465–474. doi: 10.1016/0022-2836(89)90494-4. [DOI] [PubMed] [Google Scholar]
  20. FREEMAN V. J. Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphtheriae. J Bacteriol. 1951 Jun;61(6):675–688. doi: 10.1128/jb.61.6.675-688.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fujii G., Choe S. H., Bennett M. J., Eisenberg D. Crystallization of diphtheria toxin. J Mol Biol. 1991 Dec 20;222(4):861–864. doi: 10.1016/0022-2836(91)90577-s. [DOI] [PubMed] [Google Scholar]
  22. Greenfield L., Bjorn M. J., Horn G., Fong D., Buck G. A., Collier R. J., Kaplan D. A. Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6853–6857. doi: 10.1073/pnas.80.22.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hope H. Cryocrystallography of biological macromolecules: a generally applicable method. Acta Crystallogr B. 1988 Feb 1;44(Pt 1):22–26. doi: 10.1107/s0108768187008632. [DOI] [PubMed] [Google Scholar]
  24. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  25. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  26. Karle I. L., Flippen-Anderson J. L., Kishore R., Balaram P. Cystine peptides. Antiparallel beta-sheet conformation of the cyclic biscystine peptide [Boc-Cys-Ala-Cys-NHCH3]2. Int J Pept Protein Res. 1989 Jul;34(1):37–41. doi: 10.1111/j.1399-3011.1989.tb01005.x. [DOI] [PubMed] [Google Scholar]
  27. Lewis P. N., Momany F. A., Scheraga H. A. Chain reversals in proteins. Biochim Biophys Acta. 1973 Apr 20;303(2):211–229. doi: 10.1016/0005-2795(73)90350-4. [DOI] [PubMed] [Google Scholar]
  28. London E. Diphtheria toxin: membrane interaction and membrane translocation. Biochim Biophys Acta. 1992 Mar 26;1113(1):25–51. doi: 10.1016/0304-4157(92)90033-7. [DOI] [PubMed] [Google Scholar]
  29. Lory S., Carroll S. F., Collier R. J. Ligand interactions of diphtheria toxin. II. Relationships between the NAD site and the P site. J Biol Chem. 1980 Dec 25;255(24):12016–12019. [PubMed] [Google Scholar]
  30. Madden D. R., Gorga J. C., Strominger J. L., Wiley D. C. The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC. Cell. 1992 Sep 18;70(6):1035–1048. doi: 10.1016/0092-8674(92)90252-8. [DOI] [PubMed] [Google Scholar]
  31. Michel A., Dirkx J. Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A. Biochim Biophys Acta. 1977 Mar 28;491(1):286–295. doi: 10.1016/0005-2795(77)90064-2. [DOI] [PubMed] [Google Scholar]
  32. Moodie S. L., Thornton J. M. A study into the effects of protein binding on nucleotide conformation. Nucleic Acids Res. 1993 Mar 25;21(6):1369–1380. doi: 10.1093/nar/21.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morris A. L., MacArthur M. W., Hutchinson E. G., Thornton J. M. Stereochemical quality of protein structure coordinates. Proteins. 1992 Apr;12(4):345–364. doi: 10.1002/prot.340120407. [DOI] [PubMed] [Google Scholar]
  34. Morris R. E., Gerstein A. S., Bonventre P. F., Saelinger C. B. Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation. Infect Immun. 1985 Dec;50(3):721–727. doi: 10.1128/iai.50.3.721-727.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Naglich J. G., Metherall J. E., Russell D. W., Eidels L. Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor. Cell. 1992 Jun 12;69(6):1051–1061. doi: 10.1016/0092-8674(92)90623-k. [DOI] [PubMed] [Google Scholar]
  37. Papini E., Santucci A., Schiavo G., Domenighini M., Neri P., Rappuoli R., Montecucco C. Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD binding site of diphtheria toxin. J Biol Chem. 1991 Feb 5;266(4):2494–2498. [PubMed] [Google Scholar]
  38. Papini E., Schiavo G., Sandoná D., Rappuoli R., Montecucco C. Histidine 21 is at the NAD+ binding site of diphtheria toxin. J Biol Chem. 1989 Jul 25;264(21):12385–12388. [PubMed] [Google Scholar]
  39. Papini E., Schiavo G., Tomasi M., Colombatti M., Rappuoli R., Montecucco C. Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication. Eur J Biochem. 1987 Dec 15;169(3):637–644. doi: 10.1111/j.1432-1033.1987.tb13655.x. [DOI] [PubMed] [Google Scholar]
  40. Pastan I., Chaudhary V., FitzGerald D. J. Recombinant toxins as novel therapeutic agents. Annu Rev Biochem. 1992;61:331–354. doi: 10.1146/annurev.bi.61.070192.001555. [DOI] [PubMed] [Google Scholar]
  41. Ponder J. W., Richards F. M. Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. J Mol Biol. 1987 Feb 20;193(4):775–791. doi: 10.1016/0022-2836(87)90358-5. [DOI] [PubMed] [Google Scholar]
  42. Proia R. L., Wray S. K., Hart D. A., Eidels L. Characterization and affinity labeling of the cationic phosphate-binding (nucleotide-binding) peptide located in the receptor-binding region of the B-fragment of diphtheria toxin. J Biol Chem. 1980 Dec 25;255(24):12025–12033. [PubMed] [Google Scholar]
  43. Richardson J. S. The anatomy and taxonomy of protein structure. Adv Protein Chem. 1981;34:167–339. doi: 10.1016/s0065-3233(08)60520-3. [DOI] [PubMed] [Google Scholar]
  44. Richmond T. J., Richards F. M. Packing of alpha-helices: geometrical constraints and contact areas. J Mol Biol. 1978 Mar 15;119(4):537–555. doi: 10.1016/0022-2836(78)90201-2. [DOI] [PubMed] [Google Scholar]
  45. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sandvig K., Olsnes S. Diphtheria toxin-induced channels in Vero cells selective for monovalent cations. J Biol Chem. 1988 Sep 5;263(25):12352–12359. [PubMed] [Google Scholar]
  47. Satow Y., Cohen G. H., Padlan E. A., Davies D. R. Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. J Mol Biol. 1986 Aug 20;190(4):593–604. doi: 10.1016/0022-2836(86)90245-7. [DOI] [PubMed] [Google Scholar]
  48. Schrauber H., Eisenhaber F., Argos P. Rotamers: to be or not to be? An analysis of amino acid side-chain conformations in globular proteins. J Mol Biol. 1993 Mar 20;230(2):592–612. doi: 10.1006/jmbi.1993.1172. [DOI] [PubMed] [Google Scholar]
  49. Seeman N. C., Rosenberg J. M., Suddath F. L., Kim J. J., Rich A. RNA double-helical fragments at atomic resolution. I. The crystal and molecular structure of sodium adenylyl-3',5'-uridine hexahydrate. J Mol Biol. 1976 Jun 14;104(1):109–144. doi: 10.1016/0022-2836(76)90005-x. [DOI] [PubMed] [Google Scholar]
  50. Sixma T. K., Pronk S. E., Kalk K. H., Wartna E. S., van Zanten B. A., Witholt B., Hol W. G. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature. 1991 May 30;351(6325):371–377. doi: 10.1038/351371a0. [DOI] [PubMed] [Google Scholar]
  51. Teeter M. M. Water structure of a hydrophobic protein at atomic resolution: Pentagon rings of water molecules in crystals of crambin. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6014–6018. doi: 10.1073/pnas.81.19.6014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Uchida T., Gill D. M., Pappenheimer A. M., Jr Mutation in the structural gene for diphtheria toxin carried by temperate phage . Nat New Biol. 1971 Sep 1;233(35):8–11. doi: 10.1038/newbio233008a0. [DOI] [PubMed] [Google Scholar]
  53. VAN DEN BERG L., ROSE D. Effect of freezing on the pH and composition of sodium and potassium phosphate solutions; the reciprocal system KH2PO4-Na2-HPO4-H2O. Arch Biochem Biophys. 1959 Apr;81(2):319–329. doi: 10.1016/0003-9861(59)90209-7. [DOI] [PubMed] [Google Scholar]
  54. Van Ness B. G., Howard J. B., Bodley J. W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem. 1980 Nov 25;255(22):10710–10716. [PubMed] [Google Scholar]
  55. Wilke M. E., Higaki J. N., Craik C. S., Fletterick R. J. Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases. J Mol Biol. 1991 Jun 5;219(3):511–523. doi: 10.1016/0022-2836(91)90190-h. [DOI] [PubMed] [Google Scholar]
  56. Wilson B. A., Reich K. A., Weinstein B. R., Collier R. J. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. Biochemistry. 1990 Sep 18;29(37):8643–8651. doi: 10.1021/bi00489a021. [DOI] [PubMed] [Google Scholar]
  57. Zhang K. Y., Eisenberg D. The three-dimensional profile method using residue preference as a continuous function of residue environment. Protein Sci. 1994 Apr;3(4):687–695. doi: 10.1002/pro.5560030416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhao J. M., London E. Localization of the active site of diphtheria toxin. Biochemistry. 1988 May 3;27(9):3398–3403. doi: 10.1021/bi00409a041. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES