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. 1995 Apr 15;307(Pt 2):527–534. doi: 10.1042/bj3070527

Long-range conformational effects of proteolytic removal of the last three residues of actin.

H Strzelecka-Gołaszewska 1, M Mossakowska 1, A Woźniak 1, J Moraczewska 1, H Nakayama 1
PMCID: PMC1136680  PMID: 7733893

Abstract

Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.

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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. Aspenström P., Schutt C. E., Lindberg U., Karlsson R. Mutations in beta-actin: influence on polymer formation and on interactions with myosin and profilin. FEBS Lett. 1993 Aug 23;329(1-2):163–170. doi: 10.1016/0014-5793(93)80215-g. [DOI] [PubMed] [Google Scholar]
  3. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  4. Brenner S. L., Korn E. D. On the mechanism of actin monomer-polymer subunit exchange at steady state. J Biol Chem. 1983 Apr 25;258(8):5013–5020. [PubMed] [Google Scholar]
  5. Carlier M. F. Actin polymerization and ATP hydrolysis. Adv Biophys. 1990;26:51–73. doi: 10.1016/0065-227x(90)90007-g. [DOI] [PubMed] [Google Scholar]
  6. Carlier M. F., Jean C., Rieger K. J., Lenfant M., Pantaloni D. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5034–5038. doi: 10.1073/pnas.90.11.5034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlier M. F., Pantaloni D., Korn E. D. The mechanisms of ATP hydrolysis accompanying the polymerization of Mg-actin and Ca-actin. J Biol Chem. 1987 Mar 5;262(7):3052–3059. [PubMed] [Google Scholar]
  8. Coué M., Korn E. D. ATP hydrolysis by the gelsolin-actin complex and at the pointed ends of gelsolin-capped filaments. J Biol Chem. 1986 Feb 5;261(4):1588–1593. [PubMed] [Google Scholar]
  9. Drewes G., Faulstich H. Cooperative effects on filament stability in actin modified at the C-terminus by substitution or truncation. Eur J Biochem. 1993 Feb 15;212(1):247–253. doi: 10.1111/j.1432-1033.1993.tb17656.x. [DOI] [PubMed] [Google Scholar]
  10. Drewes G., Faulstich H. The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization. J Biol Chem. 1990 Feb 25;265(6):3017–3021. [PubMed] [Google Scholar]
  11. Drummond D. R., Hennessey E. S., Sparrow J. C. The binding of mutant actins to profilin, ATP and DNase I. Eur J Biochem. 1992 Oct 1;209(1):171–179. doi: 10.1111/j.1432-1033.1992.tb17274.x. [DOI] [PubMed] [Google Scholar]
  12. Estes J. E., Selden L. A., Kinosian H. J., Gershman L. C. Tightly-bound divalent cation of actin. J Muscle Res Cell Motil. 1992 Jun;13(3):272–284. doi: 10.1007/BF01766455. [DOI] [PubMed] [Google Scholar]
  13. Frieden C., Lieberman D., Gilbert H. R. A fluorescent probe for conformational changes in skeletal muscle G-actin. J Biol Chem. 1980 Oct 10;255(19):8991–8993. [PubMed] [Google Scholar]
  14. Frieden C., Patane K. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP. Biochemistry. 1985 Jul 16;24(15):4192–4196. doi: 10.1021/bi00336a056. [DOI] [PubMed] [Google Scholar]
  15. Gershman L. C., Selden L. A., Estes J. E. High affinity binding of divalent cation to actin monomer is much stronger than previously reported. Biochem Biophys Res Commun. 1986 Mar 13;135(2):607–614. doi: 10.1016/0006-291x(86)90036-7. [DOI] [PubMed] [Google Scholar]
  16. Goldschmidt-Clermont P. J., Furman M. I., Wachsstock D., Safer D., Nachmias V. T., Pollard T. D. The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells. Mol Biol Cell. 1992 Sep;3(9):1015–1024. doi: 10.1091/mbc.3.9.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heintz D., Reichert A., Mihelic M., Voelter W., Faulstich H. Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9. FEBS Lett. 1993 Aug 23;329(1-2):9–12. doi: 10.1016/0014-5793(93)80181-s. [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. Houk T. W., Jr, Ue K. The measurement of actin concentration in solution: a comparison of methods. Anal Biochem. 1974 Nov;62(1):66–74. doi: 10.1016/0003-2697(74)90367-4. [DOI] [PubMed] [Google Scholar]
  20. Jacobson G. R., Rosenbusch J. P. ATP binding to a protease-resistant core of actin. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2742–2746. doi: 10.1073/pnas.73.8.2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kasprzak A. A. Myosin subfragment 1 inhibits dissociation of nucleotide and calcium from G-actin. J Biol Chem. 1993 Jun 25;268(18):13261–13266. [PubMed] [Google Scholar]
  23. Khaitlina SYu, Collins J. H., Kuznetsova I. M., Pershina V. P., Synakevich I. G., Turoverov K. K., Usmanova A. M. Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain. FEBS Lett. 1991 Feb 11;279(1):49–51. doi: 10.1016/0014-5793(91)80247-z. [DOI] [PubMed] [Google Scholar]
  24. Kinosian H. J., Selden L. A., Estes J. E., Gershman L. C. Nucleotide binding to actin. Cation dependence of nucleotide dissociation and exchange rates. J Biol Chem. 1993 Apr 25;268(12):8683–8691. [PubMed] [Google Scholar]
  25. Kodama T., Fukui K., Kometani K. The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate. J Biochem. 1986 May;99(5):1465–1472. doi: 10.1093/oxfordjournals.jbchem.a135616. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lorenz M., Popp D., Holmes K. C. Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J Mol Biol. 1993 Dec 5;234(3):826–836. doi: 10.1006/jmbi.1993.1628. [DOI] [PubMed] [Google Scholar]
  28. McLaughlin P. J., Gooch J. T., Mannherz H. G., Weeds A. G. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature. 1993 Aug 19;364(6439):685–692. doi: 10.1038/364685a0. [DOI] [PubMed] [Google Scholar]
  29. Miki M., Onuma H., Mihashi K. Interaction of actin water epsilon-ATP. FEBS Lett. 1974 Sep 15;46(1):17–19. doi: 10.1016/0014-5793(74)80324-8. [DOI] [PubMed] [Google Scholar]
  30. Mockrin S. C., Korn E. D. Acanthamoeba profilin interacts with G-actin to increase the rate of exchange of actin-bound adenosine 5'-triphosphate. Biochemistry. 1980 Nov 11;19(23):5359–5362. doi: 10.1021/bi00564a033. [DOI] [PubMed] [Google Scholar]
  31. Mossakowska M., Moraczewska J., Khaitlina S., Strzelecka-Golaszewska H. Proteolytic removal of three C-terminal residues of actin alters the monomer-monomer interactions. Biochem J. 1993 Feb 1;289(Pt 3):897–902. doi: 10.1042/bj2890897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Muhlrad A., Cheung P., Phan B. C., Miller C., Reisler E. Dynamic properties of actin. Structural changes induced by beryllium fluoride. J Biol Chem. 1994 Apr 22;269(16):11852–11858. [PubMed] [Google Scholar]
  33. Newman J., Zaner K. S., Schick K. L., Gershman L. C., Selden L. A., Kinosian H. J., Travis J. L., Estes J. E. Nucleotide exchange and rheometric studies with F-actin prepared from ATP- or ADP-monomeric actin. Biophys J. 1993 May;64(5):1559–1566. doi: 10.1016/S0006-3495(93)81525-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nishida E. Opposite effects of cofilin and profilin from porcine brain on rate of exchange of actin-bound adenosine 5'-triphosphate. Biochemistry. 1985 Feb 26;24(5):1160–1164. doi: 10.1021/bi00326a015. [DOI] [PubMed] [Google Scholar]
  35. O'Donoghue S. I., Miki M., dos Remedios C. G. Removing the two C-terminal residues of actin affects the filament structure. Arch Biochem Biophys. 1992 Feb 14;293(1):110–116. doi: 10.1016/0003-9861(92)90372-4. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Pollender J. M., Gruda J. Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry. Can J Biochem. 1979 Jan;57(1):49–55. doi: 10.1139/o79-006. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Schwyter D., Phillips M., Reisler E. Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1. Biochemistry. 1989 Jul 11;28(14):5889–5895. doi: 10.1021/bi00440a027. [DOI] [PubMed] [Google Scholar]
  40. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  41. Stournaras C., Drewes G., Blackholm H., Merkler I., Faulstich H. Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability. Biochim Biophys Acta. 1990 Jan 19;1037(1):86–91. doi: 10.1016/0167-4838(90)90105-o. [DOI] [PubMed] [Google Scholar]
  42. Strzelecka-Gołaszewska H., Moraczewska J., Khaitlina S. Y., Mossakowska M. Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion. Eur J Biochem. 1993 Feb 1;211(3):731–742. doi: 10.1111/j.1432-1033.1993.tb17603.x. [DOI] [PubMed] [Google Scholar]
  43. Tawada K., Wahl P., Auchet J. C. Study of actin and its interactions with heavy meromyosin and the regulatory proteins by the pulse fluorimetry in polarized light of a fluorescent probe attached to an actin cysteine. Eur J Biochem. 1978 Aug 1;88(2):411–419. doi: 10.1111/j.1432-1033.1978.tb12463.x. [DOI] [PubMed] [Google Scholar]
  44. Valentin-Ranc C., Carlier M. F. Evidence for the direct interaction between tightly bound divalent metal ion and ATP on actin. Binding of the lambda isomers of beta gamma-bidentate CrATP to actin. J Biol Chem. 1989 Dec 15;264(35):20871–20880. [PubMed] [Google Scholar]
  45. Wang Y. L., Taylor D. L. Exchange of 1,N6-etheno-ATP with actin-bound nucleotides as a tool for studying the steady-state exchange of subunits in F-actin solutions. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5503–5507. doi: 10.1073/pnas.78.9.5503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wegner A., Neuhaus J. M. Requirement of divalent cations for fast exchange of actin monomers and actin filament subunits. J Mol Biol. 1981 Dec 15;153(3):681–693. doi: 10.1016/0022-2836(81)90413-7. [DOI] [PubMed] [Google Scholar]

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