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NMR structure and dynamics of the chimeric protein SH3-F2

  • Structural-Functional Analysis of Biopolymers and Their Complexes
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Abstract

In order to further elucidate structural and dynamic principles of protein self-organization and protein-ligand interactions, a new chimeric protein was designed and a genetically engineered construct was created. SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker, and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamic properties of the protein were studied with high-resolution NMR. According to NMR data, the tertiary structure of the chimeric protein SH3-F2 has a topology that is typical for SH3 domains in the complex with the ligand forming polyproline type II helix located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However, the interactions of the ligand and the part of globule related to SH3 domain is not too large, because the analysis of protein dynamical characteristics points to the low amplitude, high-frequency ligand tumbling relative to the slow intramolecular motions of the main globule. The constructed chimera allows carrying out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

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Abbreviations

SH3:

Src-homologous domain 3

Src:

tyrosine kinase, contained in Rous sarcoma virus

WT-SH3:

the recombinant protein with the sequence of α-spectrin domain of the wild type; p41, decapeptide, having sequence APSYSPPPPP

PPII:

polyproline helix of the type II; S19-P20s, circular permutant of α-spectrin SH3-domain, having a breal between S19 and P20 positions and cross-linked N- and C-termini

SPCp41:

chimeric protein based on S19-P20s, with p41 sequence being linked to it through the three-ring linker (DCN)

SH3-F2:

chimeric protein based on S19-P20s, with the sequence APPLPPYSA being joined to its C-terminus Through the GG linker

NOE:

nuclear Overhauser effect

NOESY:

two-dimensional NOE spectroscopy

TOCSY:

total correlation spectroscopy

HSQC:

heteronuclear single-quantum correlation spectroscopy

PDB:

protein data bank

RMSD:

root mean-sqare deviations; m — overall protein rotational correlation time

References

  1. Musacchio A., Wilmanns M., Saraste M. 1994. Structure and function of the SH3 domain. Prog. Biophys. Mol. Biol. 61, 283–297.

    Article  CAS  PubMed  Google Scholar 

  2. Cowan-Jacob S.W. 2006. Structural biology of protein tyrosine kinases. Cell. Mol. Life Sci. 63, 2608–2625.

    Article  CAS  PubMed  Google Scholar 

  3. Gmeiner W.H., Horita D.A. 2001. Implications of SH3 domain structure and dynamics for protein regulation and drug design. Cell Biochem. Biophys. 35, 127–140.

    Article  CAS  PubMed  Google Scholar 

  4. Kay B.K., Williamson M.P., Sudol M. 2000. The importance of being proline: The interaction of proline-rich motifs in signaling proteins with their cognate domains. Faseb J. 14, 231–241.

    CAS  PubMed  Google Scholar 

  5. Mayer B.J. 2001. SH3 domains: Complexity in moderation. J. Cell Sci. 114, 1253–1263.

    CAS  PubMed  Google Scholar 

  6. Vidal M., Gigoux V., Garbay C. 2001. SH2 and SH3 domains as targets for antiproliferative agents. Crit. Rev. Oncol. Hematol. 40, 175–186.

    Article  CAS  PubMed  Google Scholar 

  7. Dalgarno D.C., Botfield M.C., Rickles R.J. 1997. SH3 domains and drug design: Ligands, structure, and biological function. Biopolymers. 43, 383–400.

    Article  CAS  PubMed  Google Scholar 

  8. Viguera A.R., Arrondo J.L., Musacchio A., Saraste M., Serrano L. 1994. Characterization of the interaction of natural proline-rich peptides with five different SH3 domains. Biochemistry. 33, 10925–10933.

    Article  CAS  PubMed  Google Scholar 

  9. Pisabarro M.T., Serrano L. 1996. Rational design of specific high-affinity peptide ligands for the Abl-SH3 domain. Biochemistry. 35, 10634–10640.

    Article  CAS  PubMed  Google Scholar 

  10. Arold S.T., Ulmer T.S., Mulhern T.D., Werner J.M., Ladbury J.E., Campbell I.D., Noble M.E. 2001. The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases. J. Biol. Chem. 276, 17199–17205.

    Article  CAS  PubMed  Google Scholar 

  11. Martin-Sierra F.M., Candel A.M., Casares S., Filimonov V.V., Martinez J.C., Conejero-Lara F. 2003. A binding event converted into a folding event. FEBS Lett. 553, 328–332.

    Article  CAS  PubMed  Google Scholar 

  12. Gushchina L.V., Gabdulkhanov A.G., Filimonov V.V. 2009. Design and structural-thermodynamic studies of chimerical protein derived from spectrin SH3-domain. Mol. Biol. (Moscow) 43, 444–452.

    Article  CAS  Google Scholar 

  13. Peranen J., Rikkonen M., Hyvonen M., Kaariainen L. 1996. T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. Anal. Biochem. 236, 371–373.

    Article  CAS  PubMed  Google Scholar 

  14. Gill S.C., von Hippel P.H. 1989. Calculation of protein extinction coefficients from amino-acid sequence data. Anal. Biochem. 182, 319–326.

    Article  CAS  PubMed  Google Scholar 

  15. Schagger H., von Jagow G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from to 100 kDa. Anal. Biochem. 166, 368–379.

    Article  CAS  PubMed  Google Scholar 

  16. Sattler M., Schleucher J., Griesinger C. 1999. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158.

    Article  CAS  Google Scholar 

  17. Keller R. 2004. The Computer Aided Resonance Assignment Tutorial. ISBN 3-85600-112-3.

  18. Markely J.L., Bax A., Arata Y., Hilbers C.W., Kaptein R., Sykes B.D., Wright P.E., Wutrich K. 1998. Recommendations for the presentation of NMR structures of proteins and nucleic acids. Pure Appl. Chem. 70, 117–142.

    Article  Google Scholar 

  19. Wishart D.S., Bigam C.G., Yao J., Abildgaard F., Dyson H.J., Oldfield E., Markley J.L., Sykes B.D. 1995. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR. 6, 135–140.

    Article  CAS  PubMed  Google Scholar 

  20. Farrow N.A., Muhandiram R., Singer A.U., Pascal S.M., Kay C.M., Gish G., Shoelson S.E., Pawson T., Forman-Kay J.D., Kay L.E. 1994. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 33, 5984–6003.

    Article  CAS  PubMed  Google Scholar 

  21. Delaglio F., Grzesiek S., Vuister G.W., Zhu G., Pfeifer J., Bax A. 1995. Nmr-pipe: A multidimensional spectral processing system based on Unix pipes. J. Biomol. NMR. 6, 277–293.

    Article  CAS  PubMed  Google Scholar 

  22. Orekhov V.Yu., Nolde D.E., Golovanov A.P., Korzhnev D.M., Arseniev A.S. 1995. Processing of heteronuclear NMR relaxation data with new software DASHA. Appl. Magn. Reson. 9, 581–588.

    Article  CAS  Google Scholar 

  23. Cornilescu G., Delaglio F., Bax A. 1999. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR. 13, 289–302.

    Article  CAS  PubMed  Google Scholar 

  24. Güntert P. 2004. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378.

    PubMed  Google Scholar 

  25. Torshin I.Y., Weber I.T., Harrison R.W. 2002. Geometric criteria of hydrogen bonds in ptoteins and indentification of bifurcated hydrogen bonds. Protein Eng. 15, 359–363.

    Article  CAS  PubMed  Google Scholar 

  26. Koradi R., Billeter M., Wuthrich K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55.

    Article  CAS  PubMed  Google Scholar 

  27. Musacchio A., Noble M., Pauptit R., Wierenga R., Saraste M. 1992. Crystal structure of a Src-homology 3 (SH3) domain. Nature. 359, 851–855.

    Article  CAS  PubMed  Google Scholar 

  28. Viguera A.-R. Serrano L., Wilmanns M. 1996. Different folding transition states may result in the same native structure. Nature Struct. Biol. 3, 874–880.

    Article  CAS  PubMed  Google Scholar 

  29. Feng S., Kasahara C., Rickles, R.J., Schreiber S.L. 1995. Specific interactions outside the proline-rich core of two casses of Src homology 3 ligands. Proc. Natl. Acad. Sci. USA. 92, 12408–12415.

    Article  CAS  PubMed  Google Scholar 

  30. Martínez J.C., Viguera A.R., Berisio R., Wilmanns M., Mateo P.L., Filimonov V.V., Serrano L. 1999. Thermodynamic analysis of alpha-spectrin SH3 and two of its circular permutants with different loop lengths: discerning the reasons for rapid folding in proteins. Biochemistry. 38, 549–559.

    Article  PubMed  Google Scholar 

  31. Privalov P.L. 1979. Stability of proteins: Small globular proteins. Adv. Protein Chem. 33, 167–241.

    Article  CAS  PubMed  Google Scholar 

  32. Cavanagh J., Fairbrother W.J., Palmer III A.G., Skelton N.J., Rance M. 2006. Protein NMR Spectroscopy: Principles and Practice, 2nd ed., Amsterdam: Academic.

    Google Scholar 

  33. Wuthrich K. 1976. NMR in Biological Research: Peptides and Proteins. Amsterdam: North Holland.

    Google Scholar 

  34. Daragan V.A., Mayo K.H. 1997. Motional model analyses of protein and peptide dynamics using C-13 and N-15 NMR relaxation. Prog. Nucl. Mag. Res. 31, 63–105.

    Article  CAS  Google Scholar 

  35. Kay L.E., Torchia D.A., Bax A. 1989. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry. 28, 8972–8979.

    Article  CAS  PubMed  Google Scholar 

  36. Lipari G., Szabo A. 1982. Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules: 2. Analysis of experimental results. J. Am. Chem. Soc. 104, 4559–4570.

    Article  CAS  Google Scholar 

  37. Clore G.M., Szabo A., Bax A., Kay L.E., Driscoll P.C., Gronenborn A.M. 1990. Deviations from the simple two-parameter model-free approach to the interpretation of nitrogen-15 nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 112, 4989–4991.

    Article  CAS  Google Scholar 

  38. Prokhorov D.A., Timchenko M.A., Kudrevatykh Yu.A., Fedyukina D.V., Gushchina L.V., Khristoforov V.S., Filimonov V.V., Kutyshenko V.P. 2008. NMR study of chimeric SH3 domain (“SHA-BERGERAC”) structure and dynamics. Russ. J. Bioorg. Chem. 34, 578–585.

    Article  CAS  Google Scholar 

  39. Kutyshenko V.P., Prokhorov D.A., Timchenko M.A., Kudrevatykh Yu.A., Gushchina L.V., Khristoforov V.S., Filimonov V.V., Uversky V.N. 2009. Solution structure and dynamics of the chimeric SH3 domains, SHH- and SHA-“Bergeracs.” Biochim. Biophys. Acta. 1794, 1813–1822.

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow region, 142290, Russia

    V. P. Kutyshenko, V. S. Khristoforov, D. A. Prokhorov, M. A. Timchenko, Yu. A. Kudrevatykh & D. V. Fedyukina

  2. Institute of Protein Research, Russian Academy of Science, Pushchino, Moscow region, 142290, Russia

    L. V. Gushchina & V. V. Filimonov

Authors
  1. V. P. Kutyshenko
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  2. L. V. Gushchina
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  3. V. S. Khristoforov
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  4. D. A. Prokhorov
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  5. M. A. Timchenko
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  6. Yu. A. Kudrevatykh
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  7. D. V. Fedyukina
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  8. V. V. Filimonov
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Correspondence to V. P. Kutyshenko.

Additional information

Original Russian Text © V.P. Kutyshenko, L.V. Gushchina, V.S. Khristoforo, D.A. Prokhorov, M.A. Timchenko, Yu.A. Kudrevatykh, D.V. Fedyukina, V.V. Filimonov, 2010, published in Molekulyarnaya Biologiya, 2010, Vol. 44, No. 6, pp. 1064–1074.

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Kutyshenko, V.P., Gushchina, L.V., Khristoforov, V.S. et al. NMR structure and dynamics of the chimeric protein SH3-F2. Mol Biol 44, 948–957 (2010). https://doi.org/10.1134/S0026893310060129

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  • DOI: https://doi.org/10.1134/S0026893310060129

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