Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jul 1;109(1):225–234. doi: 10.1083/jcb.109.1.225

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure

PMCID: PMC2115473  PMID: 2745549

Abstract

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Aebi U., Cohn J., Buhle L., Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986 Oct 9;323(6088):560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
  2. Aebi U., Fowler W. E., Rew P., Sun T. T. The fibrillar substructure of keratin filaments unraveled. J Cell Biol. 1983 Oct;97(4):1131–1143. doi: 10.1083/jcb.97.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahmadi B., Speakman P. T. Suberimidate crosslinking shows that a rod-shaped, low cystine, high helix protein prepared by limited proteolysis of reduced wool has four protein chains. FEBS Lett. 1978 Oct 15;94(2):365–367. doi: 10.1016/0014-5793(78)80978-8. [DOI] [PubMed] [Google Scholar]
  4. Albers K., Fuchs E. The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J Cell Biol. 1987 Aug;105(2):791–806. doi: 10.1083/jcb.105.2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bader B. L., Magin T. M., Hatzfeld M., Franke W. W. Amino acid sequence and gene organization of cytokeratin no. 19, an exceptional tail-less intermediate filament protein. EMBO J. 1986 Aug;5(8):1865–1875. doi: 10.1002/j.1460-2075.1986.tb04438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blikstad I., Lazarides E. Vimentin filaments are assembled from a soluble precursor in avian erythroid cells. J Cell Biol. 1983 Jun;96(6):1803–1808. doi: 10.1083/jcb.96.6.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caspar D. L., Cohen C., Longley W. Tropomyosin: crystal structure, polymorphism and molecular interactions. J Mol Biol. 1969 Apr 14;41(1):87–107. doi: 10.1016/0022-2836(69)90128-4. [DOI] [PubMed] [Google Scholar]
  8. Cohen C., Longley W. Tropomyosin paracrystals formed by divalent cations. Science. 1966 May 6;152(3723):794–796. doi: 10.1126/science.152.3723.794. [DOI] [PubMed] [Google Scholar]
  9. Cohen C., Lowey S., Harrison R. G., Kendrick-Jones J., Szent-Gyorgyi A. G. Segments from myosin rods. J Mol Biol. 1970 Feb 14;47(3):605–609. doi: 10.1016/0022-2836(70)90329-3. [DOI] [PubMed] [Google Scholar]
  10. Cohen C., Szent-Györgyi A. G., Kendrick-Jones J. Paramyosin and the filaments of molluscan "catch" muscles. I. Paramyosin: structure and assembly. J Mol Biol. 1971 Mar 14;56(2):223–227. doi: 10.1016/0022-2836(71)90461-x. [DOI] [PubMed] [Google Scholar]
  11. Davis J. S. Assembly processes in vertebrate skeletal thick filament formation. Annu Rev Biophys Biophys Chem. 1988;17:217–239. doi: 10.1146/annurev.bb.17.060188.001245. [DOI] [PubMed] [Google Scholar]
  12. Fraser R. D., MacRae T. P. Intermediate filament structure. Biosci Rep. 1985 Jul;5(7):573–579. doi: 10.1007/BF01117070. [DOI] [PubMed] [Google Scholar]
  13. Geisler N., Kaufmann E., Weber K. Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments. J Mol Biol. 1985 Mar 5;182(1):173–177. doi: 10.1016/0022-2836(85)90035-x. [DOI] [PubMed] [Google Scholar]
  14. Geisler N., Kaufmann E., Weber K. Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments. Cell. 1982 Aug;30(1):277–286. doi: 10.1016/0092-8674(82)90033-2. [DOI] [PubMed] [Google Scholar]
  15. Geisler N., Plessmann U., Weber K. Related amino acid sequences in neurofilaments and non-neural intermediate filaments. Nature. 1982 Apr 1;296(5856):448–450. doi: 10.1038/296448a0. [DOI] [PubMed] [Google Scholar]
  16. Geisler N., Weber K. The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins. EMBO J. 1982;1(12):1649–1656. doi: 10.1002/j.1460-2075.1982.tb01368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Georgatos S. D., Blobel G. Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments. J Cell Biol. 1987 Jul;105(1):105–115. doi: 10.1083/jcb.105.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldman A. E., Maul G., Steinert P. M., Yang H. Y., Goldman R. D. Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3839–3843. doi: 10.1073/pnas.83.11.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gruen L. C., Woods E. F. Structural studies on the microfibrillar proteins of wool. Interaction between alpha-helical segments and reassembly of a four-chain structure. Biochem J. 1983 Mar 1;209(3):587–595. doi: 10.1042/bj2090587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henderson D., Geisler N., Weber K. A periodic ultrastructure in intermediate filaments. J Mol Biol. 1982 Feb 25;155(2):173–176. doi: 10.1016/0022-2836(82)90444-2. [DOI] [PubMed] [Google Scholar]
  21. Ip W., Hartzer M. K., Pang Y. Y., Robson R. M. Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments. J Mol Biol. 1985 Jun 5;183(3):365–375. doi: 10.1016/0022-2836(85)90007-5. [DOI] [PubMed] [Google Scholar]
  22. Kallman F., Wessells N. K. Periodic repeat units of epithelial cell tonofilaments. J Cell Biol. 1967 Jan;32(1):227–231. doi: 10.1083/jcb.32.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaufmann E., Weber K., Geisler N. Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. J Mol Biol. 1985 Oct 20;185(4):733–742. doi: 10.1016/0022-2836(85)90058-0. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lewis S. A., Balcarek J. M., Krek V., Shelanski M., Cowan N. J. Sequence of a cDNA clone encoding mouse glial fibrillary acidic protein: structural conservation of intermediate filaments. Proc Natl Acad Sci U S A. 1984 May;81(9):2743–2746. doi: 10.1073/pnas.81.9.2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McLachlan A. D., Stewart M. Periodic charge distribution in the intermediate filament proteins desmin and vimentin. J Mol Biol. 1982 Dec 15;162(3):693–698. doi: 10.1016/0022-2836(82)90396-5. [DOI] [PubMed] [Google Scholar]
  27. McLachlan A. D., Stewart M. Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. J Mol Biol. 1975 Oct 25;98(2):293–304. doi: 10.1016/s0022-2836(75)80119-7. [DOI] [PubMed] [Google Scholar]
  28. Milam L., Erickson H. P. Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments. J Cell Biol. 1982 Sep;94(3):592–596. doi: 10.1083/jcb.94.3.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  30. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  31. Potschka M. The structure of intermediate filaments. Biophys J. 1986 Jan;49(1):129–130. doi: 10.1016/S0006-3495(86)83621-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Quinlan R. A., Cohlberg J. A., Schiller D. L., Hatzfeld M., Franke W. W. Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J Mol Biol. 1984 Sep 15;178(2):365–388. doi: 10.1016/0022-2836(84)90149-9. [DOI] [PubMed] [Google Scholar]
  33. Quinlan R. A., Franke W. W. Heteropolymer filaments of vimentin and desmin in vascular smooth muscle tissue and cultured baby hamster kidney cells demonstrated by chemical crosslinking. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3452–3456. doi: 10.1073/pnas.79.11.3452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Quinlan R. A., Hatzfeld M., Franke W. W., Lustig A., Schulthess T., Engel J. Characterization of dimer subunits of intermediate filament proteins. J Mol Biol. 1986 Nov 20;192(2):337–349. doi: 10.1016/0022-2836(86)90369-4. [DOI] [PubMed] [Google Scholar]
  35. Quinlan R. A., Moir R. D., Stewart M. Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation. J Cell Sci. 1989 May;93(Pt 1):71–83. doi: 10.1242/jcs.93.1.71. [DOI] [PubMed] [Google Scholar]
  36. Quinlan R. A., Stewart M. Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry. J Cell Biol. 1987 Jul;105(1):403–415. doi: 10.1083/jcb.105.1.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reisler E., Burke M., Josephs R., Harrington W. F. Crosslinking of myosin and myosin filaments. J Mechanochem Cell Motil. 1973;2(3):163–179. [PubMed] [Google Scholar]
  38. Soellner P., Quinlan R. A., Franke W. W. Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7929–7933. doi: 10.1073/pnas.82.23.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steinert P. M., Parry D. A. Intermediate filaments: conformity and diversity of expression and structure. Annu Rev Cell Biol. 1985;1:41–65. doi: 10.1146/annurev.cb.01.110185.000353. [DOI] [PubMed] [Google Scholar]
  40. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  41. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  42. Steinert P. M., Steven A. C. Splitting hairs and other intermediate filaments. 1985 Aug 29-Sep 4Nature. 316(6031):767–767. doi: 10.1038/316767a0. [DOI] [PubMed] [Google Scholar]
  43. Steinert P. M. Structure of the three-chain unit of the bovine epidermal keratin filament. J Mol Biol. 1978 Jul 25;123(1):49–70. doi: 10.1016/0022-2836(78)90376-5. [DOI] [PubMed] [Google Scholar]
  44. Stewart M. Structures of alpha-tropomyosin magnesium paracrystals. II. Stimulation of staining patterns from the sequence and some observations on the mechanism of positive staining. J Mol Biol. 1981 Jun 5;148(4):411–425. doi: 10.1016/0022-2836(81)90184-4. [DOI] [PubMed] [Google Scholar]
  45. Stromer M. H., Huiatt T. W., Richardson R. L., Robson R. M. Disassembly of synthetic 10-nm desmin filaments from smooth muscle into protofilaments. Eur J Cell Biol. 1981 Aug;25(1):136–143. [PubMed] [Google Scholar]
  46. Vikstrom K. L., Borisy G. G., Goldman R. D. Dynamic aspects of intermediate filament networks in BHK-21 cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):549–553. doi: 10.1073/pnas.86.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Woods E. F., Gruen L. C. Structural studies on the microfibrillar proteins of wool: characterization of the alpha-helix-rich particle produced by chymotryptic digestion. Aust J Biol Sci. 1981;34(5-6):515–526. doi: 10.1071/bi9810515. [DOI] [PubMed] [Google Scholar]
  48. Woods E. F. The number of polypeptide chains in the rod domain of bovine epidermal keratin. Biochem Int. 1983 Dec;7(6):769–774. [PubMed] [Google Scholar]
  49. Zackroff R. V., Goldman A. E., Jones J. C., Steinert P. M., Goldman R. D. Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology. J Cell Biol. 1984 Apr;98(4):1231–1237. doi: 10.1083/jcb.98.4.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES