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. 1993 Dec;175(23):7652–7657. doi: 10.1128/jb.175.23.7652-7657.1993

Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.

J E Haddad 1, M P Jackson 1
PMCID: PMC206922  PMID: 8244934

Abstract

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boodhoo A., Read R. J., Brunton J. Crystallization and preliminary X-ray crystallographic analysis of verotoxin-1 B-subunit. J Mol Biol. 1991 Oct 5;221(3):729–731. doi: 10.1016/0022-2836(91)80166-r. [DOI] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Fishman P. H., Moss J., Osborne J. C., Jr Interaction of choleragen with the oligosaccharide of ganglioside GM1: evidence for multiple oligosaccharide binding sites. Biochemistry. 1978 Feb 21;17(4):711–716. doi: 10.1021/bi00597a024. [DOI] [PubMed] [Google Scholar]
  5. Frazer A. C., Curtiss R., 3rd Production, properties and utility of bacterial minicells. Curr Top Microbiol Immunol. 1975;69:1–84. doi: 10.1007/978-3-642-50112-8_1. [DOI] [PubMed] [Google Scholar]
  6. Gentry M. K., Dalrymple J. M. Quantitative microtiter cytotoxicity assay for Shigella toxin. J Clin Microbiol. 1980 Sep;12(3):361–366. doi: 10.1128/jcm.12.3.361-366.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gill D. M. The arrangement of subunits in cholera toxin. Biochemistry. 1976 Mar 23;15(6):1242–1248. doi: 10.1021/bi00651a011. [DOI] [PubMed] [Google Scholar]
  8. Griffin D. E., Gentry M. K., Brown J. E. Isolation and characterization of monoclonal antibodies to Shiga toxin. Infect Immun. 1983 Jul;41(1):430–433. doi: 10.1128/iai.41.1.430-433.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haddad J. E., al-Jaufy A. Y., Jackson M. P. Minimum domain of the Shiga toxin A subunit required for enzymatic activity. J Bacteriol. 1993 Aug;175(16):4970–4978. doi: 10.1128/jb.175.16.4970-4978.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jacewicz M., Clausen H., Nudelman E., Donohue-Rolfe A., Keusch G. T. Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide. J Exp Med. 1986 Jun 1;163(6):1391–1404. doi: 10.1084/jem.163.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson M. P., Deresiewicz R. L., Calderwood S. B. Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits. J Bacteriol. 1990 Jun;172(6):3346–3350. doi: 10.1128/jb.172.6.3346-3350.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson M. P. Structure-function analyses of Shiga toxin and the Shiga-like toxins. Microb Pathog. 1990 Apr;8(4):235–242. doi: 10.1016/0882-4010(90)90050-z. [DOI] [PubMed] [Google Scholar]
  13. Jackson M. P., Wadolkowski E. A., Weinstein D. L., Holmes R. K., O'Brien A. D. Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis. J Bacteriol. 1990 Feb;172(2):653–658. doi: 10.1128/jb.172.2.653-658.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaplan S., Stretton A. O., Brenner S. Amber suppressors: efficiency of chain propagation and suppressor specific amino acids. J Mol Biol. 1965 Dec;14(2):528–533. doi: 10.1016/s0022-2836(65)80202-9. [DOI] [PubMed] [Google Scholar]
  15. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  16. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  17. Newland J. W., Strockbine N. A., Miller S. F., O'Brien A. D., Holmes R. K. Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli. Science. 1985 Oct 11;230(4722):179–181. doi: 10.1126/science.2994228. [DOI] [PubMed] [Google Scholar]
  18. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Brien A. D., Laveck G. D. Immunochemical and cytotoxic activities of Shigella dysenteriae 1 (shiga) and shiga-like toxins. Infect Immun. 1982 Mar;35(3):1151–1154. doi: 10.1128/iai.35.3.1151-1154.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Olsnes S., Eiklid K. Isolation and characterization of Shigella shigae cytotoxin. J Biol Chem. 1980 Jan 10;255(1):284–289. [PubMed] [Google Scholar]
  21. Saleh M. T., Gariépy J. Local conformational change in the B-subunit of Shiga-like toxin 1 at endosomal pH. Biochemistry. 1993 Jan 26;32(3):918–922. doi: 10.1021/bi00054a024. [DOI] [PubMed] [Google Scholar]
  22. Sandvig K., Prydz K., Ryd M., van Deurs B. Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells. J Cell Biol. 1991 May;113(3):553–562. doi: 10.1083/jcb.113.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Sixma T. K., Stein P. E., Hol W. G., Read R. J. Comparison of the B-pentamers of heat-labile enterotoxin and verotoxin-1: two structures with remarkable similarity and dissimilarity. Biochemistry. 1993 Jan 12;32(1):191–198. doi: 10.1021/bi00052a025. [DOI] [PubMed] [Google Scholar]
  26. Stein P. E., Boodhoo A., Tyrrell G. J., Brunton J. L., Read R. J. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli. Nature. 1992 Feb 20;355(6362):748–750. doi: 10.1038/355748a0. [DOI] [PubMed] [Google Scholar]
  27. 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 Mar;170(3):1116–1122. doi: 10.1128/jb.170.3.1116-1122.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Strockbine N. A., Marques L. R., Holmes R. K., O'Brien A. D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli. Infect Immun. 1985 Dec;50(3):695–700. doi: 10.1128/iai.50.3.695-700.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Surewicz W. K., Surewicz K., Mantsch H. H., Auclair F. Interaction of Shigella toxin with globotriaosyl ceramide receptor-containing membranes: a fluorescence study. Biochem Biophys Res Commun. 1989 Apr 14;160(1):126–132. doi: 10.1016/0006-291x(89)91630-6. [DOI] [PubMed] [Google Scholar]
  30. Weinstein D. L., Jackson M. P., Samuel J. E., Holmes R. K., O'Brien A. D. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol. 1988 Sep;170(9):4223–4230. doi: 10.1128/jb.170.9.4223-4230.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  32. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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