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. 1994 May;62(5):2071–2078. doi: 10.1128/iai.62.5.2071-2078.1994

Assignment of functional domains involved in ADP-ribosylation and B-oligomer binding within the carboxyl terminus of the S1 subunit of pertussis toxin.

K M Krueger 1, J T Barbieri 1
PMCID: PMC186468  PMID: 8168972

Abstract

The roles of the carboxyl terminus of the S1 subunit (composed of 235 amino acids) of pertussis toxin in the ADP-ribosylation of transducin (Gt) and in B-oligomer binding were defined by analysis of two carboxyl-terminal deletion mutants of the recombinant S1 (rS1) subunit: C204, which is composed of amino acids 1 through 204 of S1, and C219, which is composed of amino acids 1 through 219 of S1. C204 was expressed in Escherichia coli as a stable, soluble peptide that had an apparent molecular mass of 23.4 kDa. In a linear velocity assay, the specific activity of C180 was 2% and that of C204 was 80% of the activity displayed by rS1 in catalyzing the ADP-ribosylation of Gt. In addition, C204 possessed catalytic efficiencies (kcat/Km) that were 110% at variable Gt concentrations and 40% at variable NAD concentrations of those reported for rS1. These data showed that the catalytic activity of C204 approached the activity of S1. C204 and C219 were unable to associate with the B oligomer under conditions which promoted association of rS1 with the B oligomer. Consistent with these results, mixtures of C204 or C219 with the B oligomer did not elicit a clustering phenotype in CHO cells, whereas rS1 which had associated with the B oligomer was as cytotoxic as native pertussis toxin. These data indicate that residues between 219 and 235 are important in the association of the S1 subunit with the B oligomer. These data allow the assignment of functional regions to the carboxyl terminus of S1. Residues 195 to 204 are required for optimal ADP-ribosyltransferase activity, residues 205 to 219 link the catalytic region of S1 and a B-oligomer-binding region of S1, and residues 220 to 235 are required for association of S1 with the B oligomer.

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Selected References

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  1. Antoine R., Locht C. Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin. Infect Immun. 1990 Jun;58(6):1518–1526. doi: 10.1128/iai.58.6.1518-1526.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbieri J. T., Moloney B. K., Mende-Mueller L. M. Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli. J Bacteriol. 1989 Aug;171(8):4362–4369. doi: 10.1128/jb.171.8.4362-4369.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbieri J. T., Pizza M., Cortina G., Rappuoli R. Biochemical and biological activities of recombinant S1 subunit of pertussis toxin. Infect Immun. 1990 Apr;58(4):999–1003. doi: 10.1128/iai.58.4.999-1003.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartley T. D., Whiteley D. W., Mar V. L., Burns D. L., Burnette W. N. Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8353–8357. doi: 10.1073/pnas.86.21.8353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Burns D. L., Hausman S. Z., Lindner W., Robey F. A., Manclark C. R. Structural characterization of pertussis toxin A subunit. J Biol Chem. 1987 Dec 25;262(36):17677–17682. [PubMed] [Google Scholar]
  7. Burns D. L., Manclark C. R. Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem. 1986 Mar 25;261(9):4324–4327. [PubMed] [Google Scholar]
  8. Cortina G., Barbieri J. T. Localization of a region of the S1 subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity. J Biol Chem. 1991 Feb 15;266(5):3022–3030. [PubMed] [Google Scholar]
  9. Cortina G., Krueger K. M., Barbieri J. T. The carboxyl terminus of the S1 subunit of pertussis toxin confers high affinity binding to transducin. J Biol Chem. 1991 Dec 15;266(35):23810–23814. [PubMed] [Google Scholar]
  10. Giulian G. G., Moss R. L., Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem. 1983 Mar;129(2):277–287. doi: 10.1016/0003-2697(83)90551-1. [DOI] [PubMed] [Google Scholar]
  11. Hewlett E. L., Sauer K. T., Myers G. A., Cowell J. L., Guerrant R. L. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun. 1983 Jun;40(3):1198–1203. doi: 10.1128/iai.40.3.1198-1203.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  13. Kaslow H. R., Lim L. K., Moss J., Lesikar D. D. Structure-activity analysis of the activation of pertussis toxin. Biochemistry. 1987 Jan 13;26(1):123–127. doi: 10.1021/bi00375a018. [DOI] [PubMed] [Google Scholar]
  14. Kaslow H. R., Schlotterbeck J. D., Mar V. L., Burnette W. N. Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. J Biol Chem. 1989 Apr 15;264(11):6386–6390. [PubMed] [Google Scholar]
  15. Krueger K. M., Barbieri J. T. Molecular characterization of the in vitro activation of pertussis toxin by ATP. J Biol Chem. 1993 Jun 15;268(17):12570–12578. [PubMed] [Google Scholar]
  16. Krueger K. M., Mende-Mueller L. M., Barbieri J. T. Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit. J Biol Chem. 1991 May 5;266(13):8122–8128. [PubMed] [Google Scholar]
  17. Lim L. K., Sekura R. D., Kaslow H. R. Adenine nucleotides directly stimulate pertussis toxin. J Biol Chem. 1985 Mar 10;260(5):2585–2588. [PubMed] [Google Scholar]
  18. Locht C., Lobet Y., Feron C., Cieplak W., Keith J. M. The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis. J Biol Chem. 1990 Mar 15;265(8):4552–4559. [PubMed] [Google Scholar]
  19. Mattera R., Codina J., Sekura R. D., Birnbaumer L. The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase. J Biol Chem. 1986 Aug 25;261(24):11173–11179. [PubMed] [Google Scholar]
  20. Moss J., Stanley S. J., Burns D. L., Hsia J. A., Yost D. A., Myers G. A., Hewlett E. L. Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). J Biol Chem. 1983 Oct 10;258(19):11879–11882. [PubMed] [Google Scholar]
  21. Moss J., Stanley S. J., Watkins P. A., Burns D. L., Manclark C. R., Kaslow H. R., Hewlett E. L. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents. Biochemistry. 1986 May 6;25(9):2720–2725. doi: 10.1021/bi00357a066. [DOI] [PubMed] [Google Scholar]
  22. O'Keefe D. O., Cabiaux V., Choe S., Eisenberg D., Collier R. J. pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6202–6206. doi: 10.1073/pnas.89.13.6202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Stein P. E., Boodhoo A., Armstrong G. D., Cockle S. A., Klein M. H., Read R. J. The crystal structure of pertussis toxin. Structure. 1994 Jan 15;2(1):45–57. doi: 10.1016/s0969-2126(00)00007-1. [DOI] [PubMed] [Google Scholar]
  25. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  26. Tamura M., Nogimori K., Yajima M., Ase K., Ui M. A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells. J Biol Chem. 1983 Jun 10;258(11):6756–6761. [PubMed] [Google Scholar]
  27. Watkins P. A., Burns D. L., Kanaho Y., Liu T. Y., Hewlett E. L., Moss J. ADP-ribosylation of transducin by pertussis toxin. J Biol Chem. 1985 Nov 5;260(25):13478–13482. [PubMed] [Google Scholar]

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