Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1994 Feb;66(2 Pt 1):276–285. doi: 10.1016/s0006-3495(94)80791-x

Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

A Orlova 1, X Yu 1, E H Egelman 1
PMCID: PMC1275692  PMID: 8161679

Abstract

We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities.

Full text

PDF
276

Images in this article

277FIGURE 1
on p.277
280FIGURE 5
on p.280
281FIGURE 7
on p.281
283FIGURE 10
on p.283

Selected References

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

  1. Aspenström P., Karlsson R. Interference with myosin subfragment-1 binding by site-directed mutagenesis of actin. Eur J Biochem. 1991 Aug 15;200(1):35–41. doi: 10.1111/j.1432-1033.1991.tb21045.x. [DOI] [PubMed] [Google Scholar]
  2. Bremer A., Millonig R. C., Sütterlin R., Engel A., Pollard T. D., Aebi U. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model. J Cell Biol. 1991 Nov;115(3):689–703. doi: 10.1083/jcb.115.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bulinski J. C., Kumar S., Titani K., Hauschka S. D. Peptide antibody specific for the amino terminus of skeletal muscle alpha-actin. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1506–1510. doi: 10.1073/pnas.80.6.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen T., Applegate D., Reisler E. Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products. Biochemistry. 1985 Jan 1;24(1):137–144. doi: 10.1021/bi00322a019. [DOI] [PubMed] [Google Scholar]
  5. Cook R. K., Blake W. T., Rubenstein P. A. Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. J Biol Chem. 1992 May 5;267(13):9430–9436. [PubMed] [Google Scholar]
  6. Cook R. K., Root D., Miller C., Reisler E., Rubenstein P. A. Enhanced stimulation of myosin subfragment 1 ATPase activity by addition of negatively charged residues to the yeast actin NH2 terminus. J Biol Chem. 1993 Feb 5;268(4):2410–2415. [PubMed] [Google Scholar]
  7. Craig R., Szent-Györgyi A. G., Beese L., Flicker P., Vibert P., Cohen C. Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin. J Mol Biol. 1980 Jun 15;140(1):35–55. doi: 10.1016/0022-2836(80)90355-1. [DOI] [PubMed] [Google Scholar]
  8. DasGupta G., Reisler E. Actomyosin interactions in the presence of ATP and the N-terminal segment of actin. Biochemistry. 1992 Feb 18;31(6):1836–1841. doi: 10.1021/bi00121a036. [DOI] [PubMed] [Google Scholar]
  9. DasGupta G., Reisler E. Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP. J Mol Biol. 1989 Jun 20;207(4):833–836. doi: 10.1016/0022-2836(89)90249-0. [DOI] [PubMed] [Google Scholar]
  10. DasGupta G., Reisler E. Nucleotide-induced changes in the interaction of myosin subfragment 1 with actin: detection by antibodies against the N-terminal segment of actin. Biochemistry. 1991 Oct 15;30(41):9961–9966. doi: 10.1021/bi00105a021. [DOI] [PubMed] [Google Scholar]
  11. Egelman E. H. An algorithm for straightening images of curved filamentous structures. Ultramicroscopy. 1986;19(4):367–373. doi: 10.1016/0304-3991(86)90096-3. [DOI] [PubMed] [Google Scholar]
  12. Egelman E. H., DeRosier D. J. Image analysis shows that variations in actin crossover spacings are random, not compensatory. Biophys J. 1992 Nov;63(5):1299–1305. doi: 10.1016/S0006-3495(92)81716-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Egelman E. H., Francis N., DeRosier D. J. F-actin is a helix with a random variable twist. Nature. 1982 Jul 8;298(5870):131–135. doi: 10.1038/298131a0. [DOI] [PubMed] [Google Scholar]
  14. Egelman E. H., Yu X. The location of DNA in RecA-DNA helical filaments. Science. 1989 Jul 28;245(4916):404–407. doi: 10.1126/science.2667137. [DOI] [PubMed] [Google Scholar]
  15. Greene L. E. Stoichiometry of actin X S-1 cross-linked complex. J Biol Chem. 1984 Jun 25;259(12):7363–7366. [PubMed] [Google Scholar]
  16. Harris L. J., Larson S. B., Hasel K. W., Day J., Greenwood A., McPherson A. The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature. 1992 Nov 26;360(6402):369–372. doi: 10.1038/360369a0. [DOI] [PubMed] [Google Scholar]
  17. Herman I. M. Actin isoforms. Curr Opin Cell Biol. 1993 Feb;5(1):48–55. doi: 10.1016/s0955-0674(05)80007-9. [DOI] [PubMed] [Google Scholar]
  18. Holmes K. C., Popp D., Gebhard W., Kabsch W. Atomic model of the actin filament. Nature. 1990 Sep 6;347(6288):44–49. doi: 10.1038/347044a0. [DOI] [PubMed] [Google Scholar]
  19. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  20. Miller L., Kalnoski M., Yunossi Z., Bulinski J. C., Reisler E. Antibodies directed against N-terminal residues on actin do not block acto-myosin binding. Biochemistry. 1987 Sep 22;26(19):6064–6070. doi: 10.1021/bi00393a018. [DOI] [PubMed] [Google Scholar]
  21. Milligan R. A., Flicker P. F. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J Cell Biol. 1987 Jul;105(1):29–39. doi: 10.1083/jcb.105.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Orlova A., Egelman E. H. A conformational change in the actin subunit can change the flexibility of the actin filament. J Mol Biol. 1993 Jul 20;232(2):334–341. doi: 10.1006/jmbi.1993.1393. [DOI] [PubMed] [Google Scholar]
  23. Orlova A., Egelman E. H. Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis. J Mol Biol. 1992 Oct 20;227(4):1043–1053. doi: 10.1016/0022-2836(92)90520-t. [DOI] [PubMed] [Google Scholar]
  24. Prasad B. V., Burns J. W., Marietta E., Estes M. K., Chiu W. Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy. Nature. 1990 Feb 1;343(6257):476–479. doi: 10.1038/343476a0. [DOI] [PubMed] [Google Scholar]
  25. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  26. Schutt C. E., Myslik J. C., Rozycki M. D., Goonesekere N. C., Lindberg U. The structure of crystalline profilin-beta-actin. Nature. 1993 Oct 28;365(6449):810–816. doi: 10.1038/365810a0. [DOI] [PubMed] [Google Scholar]
  27. Smith T. J., Olson N. H., Cheng R. H., Liu H., Chase E. S., Lee W. M., Leippe D. M., Mosser A. G., Rueckert R. R., Baker T. S. Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody. J Virol. 1993 Mar;67(3):1148–1158. doi: 10.1128/jvi.67.3.1148-1158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stokes D. L., DeRosier D. J. The variable twist of actin and its modulation by actin-binding proteins. J Cell Biol. 1987 Apr;104(4):1005–1017. doi: 10.1083/jcb.104.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sutoh K., Ando M., Sutoh K., Toyoshima Y. Y. Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7711–7714. doi: 10.1073/pnas.88.17.7711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sutoh K. Identification of myosin-binding sites on the actin sequence. Biochemistry. 1982 Jul 20;21(15):3654–3661. doi: 10.1021/bi00258a020. [DOI] [PubMed] [Google Scholar]
  31. Sutoh K. Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry. 1983 Mar 29;22(7):1579–1585. doi: 10.1021/bi00276a009. [DOI] [PubMed] [Google Scholar]
  32. Sönnichsen F. D., Van Eyk J. E., Hodges R. S., Sykes B. D. Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide. Biochemistry. 1992 Sep 22;31(37):8790–8798. doi: 10.1021/bi00152a015. [DOI] [PubMed] [Google Scholar]
  33. Taylor K. A., Crowther R. A. 3D reconstruction from the Fourier transform of a single superlattice image of an oblique section. Ultramicroscopy. 1992 Apr-May;41(1-3):153–167. doi: 10.1016/0304-3991(92)90105-s. [DOI] [PubMed] [Google Scholar]
  34. Wang G. J., Porta C., Chen Z. G., Baker T. S., Johnson J. E. Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography. Nature. 1992 Jan 16;355(6357):275–278. doi: 10.1038/355275a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wrigley N. G., Brown E. B., Skehel J. J. Electron microscopic evidence for the axial rotation and inter-domain flexibility of the Fab regions of immunoglobulin G. J Mol Biol. 1983 Sep 25;169(3):771–774. doi: 10.1016/s0022-2836(83)80170-3. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES