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. 2000 Jul;79(1):460–467. doi: 10.1016/S0006-3495(00)76307-7

Is SH1-SH2-cross-linked myosin subfragment 1 a structural analog of the weakly-bound state of myosin?

A A Bobkov 1, E Reisler 1
PMCID: PMC1300949  PMID: 10866971

Abstract

Myosin subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.

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Selected References

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  1. Bobkov A. A., Khvorov N. V., Golitsina N. L., Levitsky D. I. Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride. FEBS Lett. 1993 Oct 11;332(1-2):64–66. doi: 10.1016/0014-5793(93)80485-d. [DOI] [PubMed] [Google Scholar]
  2. Bobkov A. A., Levitsky D. I. Differential scanning calorimetric study of the complexes of myosin subfragment 1 with nucleoside diphosphates and vanadate or beryllium fluoride. Biochemistry. 1995 Aug 1;34(30):9708–9713. doi: 10.1021/bi00030a008. [DOI] [PubMed] [Google Scholar]
  3. Bobkov A. A., Sutoh K., Reisler E. Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin. J Muscle Res Cell Motil. 1997 Oct;18(5):563–571. doi: 10.1023/a:1018667319386. [DOI] [PubMed] [Google Scholar]
  4. Bobkova E. A., Bobkov A. A., Levitsky D. I., Reisler E. Effects of SH1 and SH2 modifications on myosin: similarities and differences. Biophys J. 1999 Feb;76(2):1001–1007. doi: 10.1016/S0006-3495(99)77264-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Burke M., Reisler F., Harrington W. F. Effect of bridging the two essential thiols of myosin on its spectral and actin-binding properties. Biochemistry. 1976 May 4;15(9):1923–1927. doi: 10.1021/bi00654a020. [DOI] [PubMed] [Google Scholar]
  7. Chalovich J. M., Greene L. E., Eisenberg E. Crosslinked myosin subfragment 1: a stable analogue of the subfragment-1.ATP complex. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4909–4913. doi: 10.1073/pnas.80.16.4909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaussepied P., Mornet D., Kassab R. Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2037–2041. doi: 10.1073/pnas.83.7.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dominguez R., Freyzon Y., Trybus K. M., Cohen C. Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell. 1998 Sep 4;94(5):559–571. doi: 10.1016/s0092-8674(00)81598-6. [DOI] [PubMed] [Google Scholar]
  10. Fisher A. J., Smith C. A., Thoden J. B., Smith R., Sutoh K., Holden H. M., Rayment I. X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. Biochemistry. 1995 Jul 18;34(28):8960–8972. doi: 10.1021/bi00028a004. [DOI] [PubMed] [Google Scholar]
  11. Godfrey J. E., Harrington W. F. Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment. Biochemistry. 1970 Feb 17;9(4):886–893. doi: 10.1021/bi00806a025. [DOI] [PubMed] [Google Scholar]
  12. Golitsina N. L., Bobkov A. A., Dedova I. V., Pavlov D. A., Nikolaeva O. P., Orlov V. N., Levitsky D. I. Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride. J Muscle Res Cell Motil. 1996 Aug;17(4):475–485. doi: 10.1007/BF00123363. [DOI] [PubMed] [Google Scholar]
  13. Goodno C. C., Taylor E. W. Inhibition of actomyosin ATPase by vanadate. Proc Natl Acad Sci U S A. 1982 Jan;79(1):21–25. doi: 10.1073/pnas.79.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gulick A. M., Bauer C. B., Thoden J. B., Rayment I. X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain. Biochemistry. 1997 Sep 30;36(39):11619–11628. doi: 10.1021/bi9712596. [DOI] [PubMed] [Google Scholar]
  15. Hiratsuka T. Nucleotide-induced closure of the ATP-binding pocket in myosin subfragment-1. J Biol Chem. 1994 Nov 4;269(44):27251–27257. [PubMed] [Google Scholar]
  16. Hiratsuka Y., Eto M., Yazawa M., Morita F. Reactivities of Cys707 (SH1) in intermediate states of myosin subfragment-1 ATPase. J Biochem. 1998 Sep;124(3):609–614. doi: 10.1093/oxfordjournals.jbchem.a022155. [DOI] [PubMed] [Google Scholar]
  17. Houdusse A., Kalabokis V. N., Himmel D., Szent-Györgyi A. G., Cohen C. Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. Cell. 1999 May 14;97(4):459–470. doi: 10.1016/s0092-8674(00)80756-4. [DOI] [PubMed] [Google Scholar]
  18. Kirshenbaum K., Papp S., Highsmith S. Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions. Biophys J. 1993 Sep;65(3):1121–1129. doi: 10.1016/S0006-3495(93)81162-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levitsky D. I., Nikolaeva O. P., Orlov V. N., Pavlov D. A., Ponomarev M. A., Rostkova E. V. Differential scanning calorimetric studies on myosin and actin. Biochemistry (Mosc) 1998 Mar;63(3):322–333. [PubMed] [Google Scholar]
  20. Levitsky D. I., Ponomarev M. A., Geeves M. A., Shnyrov V. L., Manstein D. J. Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II. Eur J Biochem. 1998 Jan 15;251(1-2):275–280. doi: 10.1046/j.1432-1327.1998.2510275.x. [DOI] [PubMed] [Google Scholar]
  21. Levitsky D. I., Shnyrov V. L., Khvorov N. V., Bukatina A. E., Vedenkina N. S., Permyakov E. A., Nikolaeva O. P., Poglazov B. F. Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1. Eur J Biochem. 1992 Nov 1;209(3):829–835. doi: 10.1111/j.1432-1033.1992.tb17354.x. [DOI] [PubMed] [Google Scholar]
  22. Ma Y. Z., Taylor E. W. Kinetic mechanism of myofibril ATPase. Biophys J. 1994 May;66(5):1542–1553. doi: 10.1016/S0006-3495(94)80945-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maruta S., Henry G. D., Sykes B. D., Ikebe M. Formation of the stable myosin-ADP-aluminum fluoride and myosin-ADP-beryllium fluoride complexes and their analysis using 19F NMR. J Biol Chem. 1993 Apr 5;268(10):7093–7100. [PubMed] [Google Scholar]
  24. Phan B. C., Faller L. D., Reisler E. Kinetic and equilibrium analysis of the interactions of actomyosin subfragment-1.ADP with beryllium fluoride. Biochemistry. 1993 Aug 3;32(30):7712–7719. doi: 10.1021/bi00081a016. [DOI] [PubMed] [Google Scholar]
  25. Phan B. C., Peyser Y. M., Reisler E., Muhlrad A. Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1. Eur J Biochem. 1997 Feb 1;243(3):636–642. doi: 10.1111/j.1432-1033.1997.00636.x. [DOI] [PubMed] [Google Scholar]
  26. Ponomarev M. A., Timofeev V. P., Levitsky D. I. The difference between ADP-beryllium fluoride and ADP-aluminium fluoride complexes of the spin-labeled myosin subfragment 1. FEBS Lett. 1995 Sep 11;371(3):261–263. doi: 10.1016/0014-5793(95)00898-j. [DOI] [PubMed] [Google Scholar]
  27. Rayment I., Rypniewski W. R., Schmidt-Bäse K., Smith R., Tomchick D. R., Benning M. M., Winkelmann D. A., Wesenberg G., Holden H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 1993 Jul 2;261(5117):50–58. doi: 10.1126/science.8316857. [DOI] [PubMed] [Google Scholar]
  28. Reisler E., Burke M., Harrington W. F. Cooperative role of two sulfhydryl groups in myosin adenosine triphosphatase. Biochemistry. 1974 May 7;13(10):2014–2022. doi: 10.1021/bi00707a003. [DOI] [PubMed] [Google Scholar]
  29. Reisler E., Burke M., Himmelfarb S., Harrington W. F. Spatial proximity of the two essential sulfhydryl groups of myosin. Biochemistry. 1974 Sep 10;13(19):3837–3840. doi: 10.1021/bi00716a001. [DOI] [PubMed] [Google Scholar]
  30. Shriver J. W., Kamath U. Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin. Biochemistry. 1990 Mar 13;29(10):2556–2564. doi: 10.1021/bi00462a018. [DOI] [PubMed] [Google Scholar]
  31. Smith C. A., Rayment I. X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry. 1996 Apr 30;35(17):5404–5417. doi: 10.1021/bi952633+. [DOI] [PubMed] [Google Scholar]
  32. Smith S. J., Eisenberg E. A comparison of the effect of vanadate on the binding of myosin-subfragment-1.ADP to actin and on actomyosin subfragment 1 ATPase activity. Eur J Biochem. 1990 Oct 5;193(1):69–73. doi: 10.1111/j.1432-1033.1990.tb19305.x. [DOI] [PubMed] [Google Scholar]
  33. Wakabayashi K., Tokunaga M., Kohno I., Sugimoto Y., Hamanaka T., Takezawa Y., Wakabayashi T., Amemiya Y. Small-angle synchrotron x-ray scattering reveals distinct shape changes of the myosin head during hydrolysis of ATP. Science. 1992 Oct 16;258(5081):443–447. doi: 10.1126/science.1411537. [DOI] [PubMed] [Google Scholar]
  34. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]
  35. Wells C., Bagshaw C. R. The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins. J Muscle Res Cell Motil. 1984 Feb;5(1):97–112. doi: 10.1007/BF00713154. [DOI] [PubMed] [Google Scholar]
  36. Wells J. A., Yount R. G. Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4966–4970. doi: 10.1073/pnas.76.10.4966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Werber M. M., Peyser Y. M., Muhlrad A. Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes. Biochemistry. 1992 Aug 11;31(31):7190–7197. doi: 10.1021/bi00146a023. [DOI] [PubMed] [Google Scholar]
  38. Xie L., Li W. X., Barnett V. A., Schoenberg M. Graphical evaluation of alkylation of myosin's SH1 and SH2: the N-phenylmaleimide reaction. Biophys J. 1997 Feb;72(2 Pt 1):858–865. doi: 10.1016/s0006-3495(97)78720-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xie L., Li W. X., Rhodes T., White H., Schoenberg M. Transient kinetic analysis of N-phenylmaleimide-reacted myosin subfragment-1. Biochemistry. 1999 May 4;38(18):5925–5931. doi: 10.1021/bi981778o. [DOI] [PubMed] [Google Scholar]
  40. Xie L., Schoenberg M. Binding of SH1-SH2-modified myosin subfragment-1 to actin. Biochemistry. 1998 Jun 2;37(22):8048–8053. doi: 10.1021/bi980319k. [DOI] [PubMed] [Google Scholar]

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