Journal of Computer-Aided Molecular Design

, Volume 31, Issue 12, pp 1063–1072 | Cite as

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

  • Josephine Alba
  • Maria Jose Marcaida
  • Jesus Prieto
  • Guillermo Montoya
  • Rafael MolinaEmail author
  • Marco D’AbramoEmail author


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.


Endonuclease I-DmoI X-ray Molecular dynamics Protein dynamics and activity Electric field 



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 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.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

10822_2017_87_MOESM1_ESM.docx (460 kb)
Supplementary material 1 (DOCX 459 KB)


  1. 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. CrossRefGoogle Scholar
  2. 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. CrossRefGoogle Scholar
  3. 3.
    Molina R et al (2012) Non-specific protein–DNA interactions control I-CreI target binding and cleavage. Nucleic Acids Res 40:6936–6945. CrossRefGoogle Scholar
  4. 4.
    Munoz IG et al (2011) Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 39:729–743. CrossRefGoogle Scholar
  5. 5.
    Paques F, Duchateau P (2007) Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7:49–66CrossRefGoogle Scholar
  6. 6.
    Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys 38:49–95. CrossRefGoogle Scholar
  7. 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. CrossRefGoogle Scholar
  8. 8.
    Molina R et al (2016) Key players in I-DmoI endonuclease catalysis revealed from structure and dynamics. ACS Chem Biol 11:1401–1407. CrossRefGoogle Scholar
  9. 9.
    Molina R et al (2015) Engineering a nickase on the homing endonuclease I-DmoI scaffold. J Biol Chem 290:18534–18544. CrossRefGoogle Scholar
  10. 10.
    Dalgaard JZ, Garrett RA, Belfort M (1993) A site-specific endonuclease encoded by a typical archaeal intron. Proc Natl Acad Sci USA 90:5414–5417CrossRefGoogle Scholar
  11. 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. CrossRefGoogle Scholar
  12. 12.
    Amadei A, Linssen AB, Berendsen HJ (1993) Essential dynamics of proteins. Proteins 17:412–425. CrossRefGoogle Scholar
  13. 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. CrossRefGoogle Scholar
  14. 14.
    Dupureur CM (2008) Roles of metal ions in nucleases. Curr Opin Chem Biol 12:250–255. CrossRefGoogle Scholar
  15. 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. CrossRefGoogle Scholar
  16. 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. CrossRefGoogle Scholar
  17. 17.
    Molina R et al (2015) Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nat Struct Mol Biol 22:65–72. CrossRefGoogle Scholar
  18. 18.
    Aragones AC et al (2016) Electrostatic catalysis of a Diels-Alder reaction. Nature 531:88–91. CrossRefGoogle Scholar
  19. 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. CrossRefGoogle Scholar
  20. 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. CrossRefGoogle Scholar
  21. 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. CrossRefGoogle Scholar
  22. 22.
    Epinat JC et al (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 31:2952–2962CrossRefGoogle Scholar
  23. 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. CrossRefGoogle Scholar
  24. 24.
    Kabsch W (2010) Xds. Acta Crystallogr D 66:125–132. CrossRefGoogle Scholar
  25. 25.
    Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D 62:72–82. CrossRefGoogle Scholar
  26. 26.
    McCoy AJ et al (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674. CrossRefGoogle Scholar
  27. 27.
    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D 66:486–501. CrossRefGoogle Scholar
  28. 28.
    Adams PD et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66:213–221. CrossRefGoogle Scholar
  29. 29.
    Hess B, Bekker H, Brendsen HJC, Fraaije J (1997) LINCS: a linear constant solver for molecular simulations. J Comput Chem 18:1463–1472Google Scholar
  30. 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 98:10089Google Scholar
  31. 31.
    Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56CrossRefGoogle Scholar
  32. 32.
    Hornak V et al (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65:712–725. CrossRefGoogle Scholar
  33. 33.
    Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. J Chem Phys 126:014101. CrossRefGoogle Scholar
  34. 34.
    Luzar A, Chandler D Hydrogen-bond kinetics in liquid water. Nature 379:55–57.

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of ChemistrySapienza University of RomeRomeItaly
  2. 2.Institute of Bioengineering, School of Life SciencesÉcole Polytechnique Fédérale de Lausanne (EPFL)LausanneSwitzerland
  3. 3.Spanish National Cancer CenterMadridSpain
  4. 4.Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark
  5. 5.Deparment of Crystallography and Structural Biology, Institute of Physical Chemistry “Rocasolano”CSICMadridSpain

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