Skip to main content
SpringerLink
Log in

Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

  • Published:
Journal of Computer-Aided Molecular Design Aims and scope Submit manuscript

Abstract

I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Chan SH, Stoddard BL, Xu SY (2011) Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity. Nucleic Acids Res 39:1–18. https://doi.org/10.1093/nar/gkq742

    Article  CAS  Google Scholar 

  2. Galetto R, Duchateau P, Paques F (2009) Targeted approaches for gene therapy and the emergence of engineered meganucleases. Expert Opin Biol Ther 9:1289–1303. https://doi.org/10.1517/14712590903213669

    Article  CAS  Google Scholar 

  3. Molina R et al (2012) Non-specific protein–DNA interactions control I-CreI target binding and cleavage. Nucleic Acids Res 40:6936–6945. https://doi.org/10.1093/nar/gks320

    Article  CAS  Google Scholar 

  4. Munoz IG et al (2011) Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 39:729–743. https://doi.org/10.1093/nar/gkq801

    Article  CAS  Google Scholar 

  5. Paques F, Duchateau P (2007) Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7:49–66

    Article  CAS  Google Scholar 

  6. Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys 38:49–95. https://doi.org/10.1017/S0033583505004063

    Article  CAS  Google Scholar 

  7. Marcaida MJ et al (2008) Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering. Proc Natl Acad Sci USA 105:16888–16893. https://doi.org/10.1073/pnas.0804795105

    Article  CAS  Google Scholar 

  8. Molina R et al (2016) Key players in I-DmoI endonuclease catalysis revealed from structure and dynamics. ACS Chem Biol 11:1401–1407. https://doi.org/10.1021/acschembio.5b00730

    Article  CAS  Google Scholar 

  9. Molina R et al (2015) Engineering a nickase on the homing endonuclease I-DmoI scaffold. J Biol Chem 290:18534–18544. https://doi.org/10.1074/jbc.M115.658666

    Article  CAS  Google Scholar 

  10. Dalgaard JZ, Garrett RA, Belfort M (1993) A site-specific endonuclease encoded by a typical archaeal intron. Proc Natl Acad Sci USA 90:5414–5417

    Article  CAS  Google Scholar 

  11. Prieto J et al (2008) Generation and analysis of mesophilic variants of the thermostable archaeal I-DmoI homing endonuclease. J Biol Chem 283:4364–4374. https://doi.org/10.1074/jbc.M706323200

    Article  CAS  Google Scholar 

  12. Amadei A, Linssen AB, Berendsen HJ (1993) Essential dynamics of proteins. Proteins 17:412–425. https://doi.org/10.1002/prot.340170408

    Article  CAS  Google Scholar 

  13. Chevalier BS, Monnat RJ, Jr. & Stoddard BL (2001) The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites. Nat Struct Biol 8:312–316. https://doi.org/10.1038/86181

    Article  CAS  Google Scholar 

  14. Dupureur CM (2008) Roles of metal ions in nucleases. Curr Opin Chem Biol 12:250–255. https://doi.org/10.1016/j.cbpa.2008.01.012

    Article  CAS  Google Scholar 

  15. Dupureur CM (2010) One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies. Metallomics 2:609–620. https://doi.org/10.1039/c0mt00013b

    Article  CAS  Google Scholar 

  16. Ivanov I, Tainer JA, McCammon JA (2007) Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV. Proc Natl Acad Sci USA 104:1465–1470. https://doi.org/10.1073/pnas.0603468104

    Article  CAS  Google Scholar 

  17. Molina R et al (2015) Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nat Struct Mol Biol 22:65–72. https://doi.org/10.1038/nsmb.2932

    Article  CAS  Google Scholar 

  18. Aragones AC et al (2016) Electrostatic catalysis of a Diels-Alder reaction. Nature 531:88–91. https://doi.org/10.1038/nature16989

    Article  CAS  Google Scholar 

  19. Amadei A, D’Alessandro M, Paci M, Di Nola A, Aschi M (2006) On the effect of a point mutation on the reactivity of CuZn superoxide dismutase: a theoretical study. J Phys Chem B 110:7538–7544. https://doi.org/10.1021/jp057095h

    Article  CAS  Google Scholar 

  20. Shaik S, de Visser SP, Kumar D (2004) External electric field will control the selectivity of enzymatic-like bond activations. J Am Chem Soc 126:11746–11749. https://doi.org/10.1021/ja047432k

    Article  CAS  Google Scholar 

  21. Arnould S et al (2006) Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol Biol 355:443–458. https://doi.org/10.1016/j.jmb.2005.10.065

    Article  CAS  Google Scholar 

  22. Epinat JC et al (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 31:2952–2962

    Article  CAS  Google Scholar 

  23. Redondo P, Prieto J, Ramos E, Blanco FJ, Montoya G (2007) Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA. Acta Crystallogr F 63:1017–1020. https://doi.org/10.1107/S1744309107049706

    Article  CAS  Google Scholar 

  24. Kabsch W (2010) Xds. Acta Crystallogr D 66:125–132. https://doi.org/10.1107/S0907444909047337

    Article  CAS  Google Scholar 

  25. Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D 62:72–82. https://doi.org/10.1107/S0907444905036693

    Article  Google Scholar 

  26. McCoy AJ et al (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674. https://doi.org/10.1107/S0021889807021206

    Article  CAS  Google Scholar 

  27. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D 66:486–501. https://doi.org/10.1107/S0907444910007493

    Article  CAS  Google Scholar 

  28. Adams PD et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66:213–221. https://doi.org/10.1107/S0907444909052925

    Article  CAS  Google Scholar 

  29. Hess B, Bekker H, Brendsen HJC, Fraaije J (1997) LINCS: a linear constant solver for molecular simulations. J Comput Chem 18:1463–1472

  30. Darden T, Tork D, Pedersen L (1997) Particle mesh Ewald: an N-log(N) method for Ewald sums in large systems. J Chem Phys 98:10089

  31. Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56

    Article  CAS  Google Scholar 

  32. Hornak V et al (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65:712–725. https://doi.org/10.1002/prot.21123

    Article  CAS  Google Scholar 

  33. Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. J Chem Phys 126:014101. https://doi.org/10.1063/1.2408420

    Article  Google Scholar 

  34. Luzar A, Chandler D Hydrogen-bond kinetics in liquid water. Nature 379:55–57. https://doi.org/10.1038/379055a0

Download references

Acknowledgements

This work was supported by Ministero dell’Istruzione, Università e Ricerca (R. Levi-Montalcini fellow to M.D.) and by Sapienza, University of Rome (Grant “Ateneo 2015”). We acknowledge CINECA Supercomputing Center, NVIDIA Academic Program, the Dept. of Chemistry for computational resources and the staffs at ALBA and SLS synchrotrons for helping in data collection.

Author information

Authors and Affiliations

  1. Department of Chemistry, Sapienza University of Rome, P.le A. Moro, 5, 00185, Rome, Italy

    Josephine Alba & Marco D’Abramo

  2. Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland

    Maria Jose Marcaida

  3. Spanish National Cancer Center, 28029, Madrid, Spain

    Jesus Prieto

  4. Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen, Denmark

    Guillermo Montoya

  5. Deparment of Crystallography and Structural Biology, Institute of Physical Chemistry “Rocasolano”, CSIC, Serrano, 119, 28006, Madrid, Spain

    Rafael Molina

Authors
  1. Josephine Alba
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Jose Marcaida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jesus Prieto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guillermo Montoya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rafael Molina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marco D’Abramo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JA performed the Molecular Dynamics simulations; MJM performed the crystallization assays and X-ray data collection; MJM and RM carried out the crystal data processing, model building and refinement; RM, MJM, JP and GM were involved in the crystallographic analysis; J.A. and R.M. prepared the figures; JA and MD analysed the trajectories; RM and MD discussed the data and wrote the manuscript.

Corresponding authors

Correspondence to Rafael Molina or Marco D’Abramo.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 459 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alba, J., Marcaida, M.J., Prieto, J. et al. Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI. J Comput Aided Mol Des 31, 1063–1072 (2017). https://doi.org/10.1007/s10822-017-0087-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10822-017-0087-5

Keywords

Search

Navigation