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Journal of Protein Chemistry

, Volume 10, Issue 1, pp 49–59 | Cite as

The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function

  • Kiran M. Khandke
  • Thomas Fairwell
  • Emory H. Braswell
  • Belur N. Manjula
Article

Abstract

Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the “dimeric form” consisted of a dimer in rapid equilibrium with its monomer, whereas the “monomeric form” does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.

Key words

Streptococcal M protein N-terminal region coiled-coil dimer/monomer molecular assembly molecular hinge 

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References

  1. Ackers, G. K. (1975). InThe Proteins (Neurath, H., and Hill, R. L., eds.), Academic Press, New York, pp. 1–94.Google Scholar
  2. Beachey, E. H., Seyer, J. M., and Kang, A. H. (1980). InStreptococcal Diseases and the Immune Response (Read, S. E., and Zabriskie, J. B., eds.), Academic Press, New York, pp. 149–160.Google Scholar
  3. Beachey, E. H., and Seyer, J. M. (1986).J. Immunol. 136, 2287–2292.Google Scholar
  4. Beachey, E. H., Seyer, J. M., and Dale, J. B. (1987).J. Exp. Med. 166, 647–656.Google Scholar
  5. Becker, C. G. (1964).Am. J. Pathol. 44, 51–60.Google Scholar
  6. Cohn, E. J., and Edsall, J. T. (1943). InProteins, Amino Acids, and Peptides, Reinhold Publishing, New York, pp. 370–381.Google Scholar
  7. Dale, J. B., Seyer, J. M., and Beachey, E. H. (1983).J. Exp. Med. 158, 1727–1723.Google Scholar
  8. Dale, J. B., and Beachey, E. H. (1986).J. Exp. Med. 163, 1191–1202.Google Scholar
  9. Fischetti, V. A., Gotschlich, E. C., Siviglia, G., and Zabriskie, J. B. (1976).J. Exp. Med. 144, 32–53.Google Scholar
  10. Fischetti, V. A., Jones, K. F., Manjula, B. N., and Scott, J. R. (1984).J. Exp. Med. 159, 1083–1095.Google Scholar
  11. Fischetti, V. A., Parry, D. A. D., Trus, B. L., Hollingshead, S. K., Scott, J. R., and Manjula, B. N. (1988).Proteins: Struct. Func. Genet. 3, 60–69.Google Scholar
  12. Fischetti, V. A. (1989).Clin. Microbiol. Rev. 2, 285–314.Google Scholar
  13. Haanes, E. J., and Cleary, P. P. (1989).J. Bacteriol. 171, 6397–6408.Google Scholar
  14. Hodges, R. S., Saund, A. K., Chong, P. C. S., St.-Pierre, S. A., and Reid, R. E. (1981).J. Biol. Chem. 256, 1214–1224.Google Scholar
  15. Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1987).Infect. Immun. 55, 3237–3239.Google Scholar
  16. Jones, K. F., Manjula, B. N., Johnston, K. H., Hollingshead, S. K., Scott, J. R., and Fischetti, V. A. (1985).J. Exp. Med. 161, 623–628.Google Scholar
  17. Jones, K. F., Khan, S. A., Erickson, B. W., Hollingshead, S. K., Scott, J. R., and Fischetti, V. A. (1986).J. Exp. Med. 164, 1226–1238.Google Scholar
  18. Jones, K. F., and Fischetti, V. A. (1988).J. Exp. Med. 167, 1114–1123.Google Scholar
  19. Khandke, K. M., Fairwell, T., and Manjula, B. N. (1987).J. Exp. Med. 166, 151–162.Google Scholar
  20. Khandke, K. M., Fairwell, T., Acharya, A. S., Trus, B. L., and Manjula, B. N. (1988).J. Biol. Chem. 263, 5075–5082.Google Scholar
  21. Khandke, K. M., Fairwell, T., Acharya, A. S., and Manjula, B. N. (1990).J. Protein Chem. 9, 511–522.Google Scholar
  22. Kraus, W., Haanes-Fritz, E., Cleary, P. P., Seyer, J. M., Dale, J. B., and Beachey, E. H. (1987).J. Immunol. 139, 3084–3090.Google Scholar
  23. Laemmli, U. K. (1970).Nature 227, 680–685.Google Scholar
  24. Lancefield, R. C. (1959).J. Exp. Med. 110, 271–292.Google Scholar
  25. Lancefield, R. C. (1962).J. Immunol. 89, 307–313.Google Scholar
  26. Manjula, B. N., and Fischetti, V. A. (1980a).J. Immunol. 124, 261–267.Google Scholar
  27. Manjula, B. N., and Fischetti, V. A. (1980b).J. Exp. Med. 151, 695–708.Google Scholar
  28. Manjula, B. N., Acharya, A. S., Mische, S. M., Fairwell, T., and Fischetti, V. A. (1984).J. Biol. Chem. 259, 3686–3693.Google Scholar
  29. Manjula, B. N., Schmidt, M. L., and Fischetti, V. A. (1985a).Infect. Immun. 50, 610–613.Google Scholar
  30. Manjula, B. N., Trus, B. L., and Fischetti, V. A. (1985b).Proc. Natl. Acad. Sci. USA 82, 1064–1068.Google Scholar
  31. Manjula, B. N., Acharya, A. S., Fairwell, T., and Fischetti, V. A. (1986).J. Exp. Med. 163, 129–138.Google Scholar
  32. Manjula, B. N. (1988).Eur. J. Epidemiol. 4, 289–300.Google Scholar
  33. McLachlan, A. D., and Stewart, M. (1975).J. Mol. Biol. 98, 393–408.Google Scholar
  34. Moravek, L., Kuhnemund, O., Havlicek, J., Kopecky, P., and Pavlik, M. (1986).FEBS Lett. 208, 435–438.Google Scholar
  35. Phillips, G. N., Flicker, P. F., Cohen, C., Manjula, B. N., and Fischetti, V. A. (1981).Proc. Natl. Acad. Sci. USA 78, 4689–4693.Google Scholar
  36. Rotta, J., Krause, R. C., Lancefield, R. C., Everly, W., and lackland, H. (1971).J. Exp. Med. 134, 1298–1315.Google Scholar
  37. Scott, J. R., Hollingshead, S. K., and Fischetti, V. A. (1986).Infect. Immun. 52, 609–612.Google Scholar
  38. Swanson, J., Hsu, K. C., and Gotschlich, E. C. (1969).J. Exp. Med. 130, 1063–1091.Google Scholar
  39. Towbin, H., Staehelin, T., and Gordon, J. (1979).Proc. Natl. Acad. Sci. USA 76, 4350–4354.Google Scholar
  40. Yphantis, D. A., and Arakawa, T. (1987).Biochemistry 26, 5422–5427.Google Scholar

Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Kiran M. Khandke
    • 1
  • Thomas Fairwell
    • 2
  • Emory H. Braswell
    • 3
  • Belur N. Manjula
    • 1
  1. 1.The Rockefeller UniversityNew York
  2. 2.National Heart, Lung and Blood InstituteNIHBethesda
  3. 3.The National Analytical Ultracentrifuge FacilityThe University of ConnecticutStorrs

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