Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The role of protein dynamics in the evolution of new enzyme function

Abstract

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Locations and effects of substitutions that accumulated across the evolutionary trajectory.
Figure 2: Intermolecular interaction networks in R0 and R22.
Figure 3: Changes in protein disorder and conformation across an evolutionary trajectory.
Figure 4: Conformational sampling of PTE-like and AE-like states by evolutionary intermediates R6 and Rev6.
Figure 5: Epistatic interactions between mutations that accumulate in R22.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Kirby, A.J. & Hollfelder, F. From Enzyme Models to Model Enzymes (Royal Society of Chemistry, 2009).

  2. Kraut, D.A., Carroll, K.S. & Herschlag, D. Challenges in enzyme mechanism and energetics. Annu. Rev. Biochem. 72, 517–571 (2003).

    CAS  PubMed  Article  Google Scholar 

  3. Ma, B. & Nussinov, R. Enzyme dynamics point to stepwise conformational selection in catalysis. Curr. Opin. Chem. Biol. 14, 652–659 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Warshel, A. Computer simulations of enzyme catalysis: methods, progress, and insights. Annu. Rev. Biophys. Biomol. Struct. 32, 425–443 (2003).

    CAS  PubMed  Article  Google Scholar 

  5. Elias, M., Wieczorek, G., Rosenne, S. & Tawfik, D.S. The universality of enzymatic rate-temperature dependency. Trends Biochem. Sci. 39, 1–7 (2014).

    CAS  PubMed  Article  Google Scholar 

  6. Bhabha, G. et al. A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science 332, 234–238 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Silva, R.G., Murkin, A.S. & Schramm, V.L. Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme. Proc. Natl. Acad. Sci. USA 108, 18661–18665 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. Bhabha, G. et al. Divergent evolution of protein conformational dynamics in dihydrofolate reductase. Nat. Struct. Mol. Biol. 20, 1243–1249 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Fraser, J.S. et al. Hidden alternative structures of proline isomerase essential for catalysis. Nature 462, 669–673 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Gobeil, S.M. et al. Maintenance of native-like protein dynamics may not be required for engineering functional proteins. Chem. Biol. 21, 1330–1340 (2014).

    CAS  PubMed  Article  Google Scholar 

  11. Jackson, C.J. et al. Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase. Proc. Natl. Acad. Sci. USA 106, 21631–21636 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. Glowacki, D.R., Harvey, J.N. & Mulholland, A.J. Taking Ockham's razor to enzyme dynamics and catalysis. Nat. Chem. 4, 169–176 (2012).

    CAS  PubMed  Article  Google Scholar 

  13. Kamerlin, S.C. & Warshel, A. At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis? Proteins 78, 1339–1375 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. O'Brien, P.J. & Hollfelder, F. Hitting a moving target?–Understanding how conformational diversity impacts enzymatic catalysis. Curr. Opin. Chem. Biol. 14, 634–635 (2010).

    CAS  PubMed  Article  Google Scholar 

  15. Fraser, J.S. & Jackson, C.J. Mining electron density for functionally relevant protein polysterism in crystal structures. Cell. Mol. Life Sci. 68, 1829–1841 (2011).

    CAS  PubMed  Article  Google Scholar 

  16. Ramanathan, A., Savol, A., Burger, V., Chennubhotla, C.S. & Agarwal, P.K. Protein conformational populations and functionally relevant substates. Acc. Chem. Res. 47, 149–156 (2014).

    CAS  PubMed  Article  Google Scholar 

  17. Klinman, J.P. & Kohen, A. Evolutionary aspects of enzyme dynamics. J. Biol. Chem. 289, 30205–30212 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Colletier, J.P. et al. Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions. Mol. Biol. Evol. 29, 1683–1694 (2012).

    CAS  PubMed  Article  Google Scholar 

  19. Tokuriki, N. & Tawfik, D.S. Protein dynamism and evolvability. Science 324, 203–207 (2009).

    CAS  PubMed  Article  Google Scholar 

  20. James, L.C. & Tawfik, D.S. Conformational diversity and protein evolution–a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 (2003).

    CAS  PubMed  Article  Google Scholar 

  21. Tokuriki, N. et al. Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme. Nat. Commun. 3, 1257 (2012).

    PubMed  Article  CAS  Google Scholar 

  22. Dellus-Gur, E. et al. Negative epistasis and evolvability in TEM-1 β-lactamase—the thin line between an enzyme's conformational freedom and disorder. J. Mol. Biol. 427, 2396–2409 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Caldwell, S.R., Newcomb, J.R., Schlecht, K.A. & Raushel, F.M. Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry 30, 7438–7444 (1991).

    CAS  PubMed  Article  Google Scholar 

  24. Kaltenbach, M., Jackson, C.J., Campbell, E.C., Hollfelder, F. & Tokuriki, N. Reverse evolution leads to genotypic incompatibility despite functional and active site convergence. eLife 4, e06492 (2015).

    PubMed Central  Article  Google Scholar 

  25. Bora, R.P., Mills, M.J., Frushicheva, M.P. & Warshel, A. On the challenge of exploring the evolutionary trajectory from phosphotriesterase to arylesterase using computer simulations. J. Phys. Chem. B 119, 3434–3445 (2015).

    CAS  PubMed  Article  Google Scholar 

  26. Doncheva, N.T., Assenov, Y., Domingues, F.S. & Albrecht, M. Topological analysis and interactive visualization of biological networks and protein structures. Nat. Protoc. 7, 670–685 (2012).

    CAS  PubMed  Article  Google Scholar 

  27. Fetics, S.K. et al. Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD. Structure 23, 505–516 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. Sethi, A., Eargle, J., Black, A.A. & Luthey-Schulten, Z. Dynamical networks in tRNA:protein complexes. Proc. Natl. Acad. Sci. USA 106, 6620–6625 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Jackson, C.J. et al. In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase. J. Mol. Biol. 375, 1189–1196 (2008).

    CAS  PubMed  Article  Google Scholar 

  30. van den Bedem, H., Bhabha, G., Yang, K., Wright, P.E. & Fraser, J.S. Automated identification of functional dynamic contact networks from X-ray crystallography. Nat. Methods 10, 896–902 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. van den Bedem, H., Dhanik, A., Latombe, J.C. & Deacon, A.M. Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers. Acta Crystallogr. D Biol. Crystallogr. 65, 1107–1117 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Burnley, B.T., Afonine, P.V., Adams, P.D. & Gros, P. Modelling dynamics in protein crystal structures by ensemble refinement. eLife 1, e00311 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. Ortlund, E.A., Bridgham, J.T., Redinbo, M.R. & Thornton, J.W. Crystal structure of an ancient protein: evolution by conformational epistasis. Science 317, 1544–1548 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Chao, F.A. et al. Structure and dynamics of a primordial catalytic fold generated by in vitro evolution. Nat. Chem. Biol. 9, 81–83 (2013).

    CAS  PubMed  Article  Google Scholar 

  35. Giger, L. et al. Evolution of a designed retro-aldolase leads to complete active site remodeling. Nat. Chem. Biol. 9, 494–498 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Khersonsky, O. et al. Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution. J. Mol. Biol. 407, 391–412 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Preiswerk, N. et al. Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase. Proc. Natl. Acad. Sci. USA 111, 8013–8018 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Sykora, J. et al. Dynamics and hydration explain failed functional transformation in dehalogenase design. Nat. Chem. Biol. 10, 428–430 (2014).

    CAS  PubMed  Article  Google Scholar 

  39. Morley, K.L. & Kazlauskas, R.J. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 23, 231–237 (2005).

    CAS  PubMed  Article  Google Scholar 

  40. Love, C.A., Lilley, P.E. & Dixon, N.E. Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene 176, 49–53 (1996).

    CAS  PubMed  Article  Google Scholar 

  41. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Collaborative, C.P. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

    Article  Google Scholar 

  43. Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Vagin, A.A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  Article  PubMed  Google Scholar 

  47. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  48. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    CAS  Article  PubMed  Google Scholar 

  49. Schmid, N. et al. Definition and testing of the GROMOS force-field versions 54A7 and 54B7. Eur. Biophys. J. 40, 843–856 (2011).

    CAS  PubMed  Article  Google Scholar 

  50. Berendsen, H., Postma, J., van Gunsteren, W.F. & Hermans, J. in Intermolecular Forces Vol. 14 (ed. Pullman, B.) 331–342 (Springer Netherlands, 1981).

  51. Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., DiNola, A. & Haak, J.R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684 (1984).

    CAS  Article  Google Scholar 

  52. Tironi, I.G., Sperb, R., Smith, P.E. & van Gunsteren, W.F. A generalized reaction field method for molecular dynamics simulations. J. Chem. Phys. 102, 5451 (1995).

    CAS  Article  Google Scholar 

  53. Heinz, T.N., van Gunsteren, W.F. & Hünenberger, P.H. Comparison of four methods to compute the dielectric permittivity of liquids from molecular dynamics simulations. J. Chem. Phys. 115, 1125 (2001).

    CAS  Article  Google Scholar 

  54. Hess, B., Bekker, H., Berendsen, H.J.C. & Fraaije, J.G.E.M. LINCS: a linear constraint solver for molecular simulations. J. Chem. Phys. 18, 1463–1472 (1997).

    CAS  Google Scholar 

  55. Miyamoto, S. & Kollman, P.A. SETTLE: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem. 13, 952–962 (1992).

    CAS  Article  Google Scholar 

  56. Bakan, A., Meireles, L.M. & Bahar, I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics 27, 1575–1577 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Grant, B.J., Rodrigues, A.P., ElSawy, K.M., McCammon, J.A. & Caves, L.S. Bio3d: an R package for the comparative analysis of protein structures. Bioinformatics 22, 2695–2696 (2006).

    CAS  PubMed  Article  Google Scholar 

  58. Van Wart, A.T., Durrant, J., Votapka, L. & Amaro, R.E. Weighted Implementation of Suboptimal Paths (WISP): An Optimized Algorithm and Tool for Dynamical Network Analysis. J. Chem. Theory Comput. 10, 511–517 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graphics 14, 33–38 (1996).

    CAS  Article  Google Scholar 

  60. Suhre, K. & Sanejouand, Y.H. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32, W610–W614 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank D.S. Tawfik for stimulating discussions. C.J.J. thanks the Australian Research Council for a Future Fellowship (FT140101059) and Discovery Project (DP130102144). This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. F.H. thanks the Biotechnology and Biological Sciences Research Council and European Research Council (starting investigator grants). M.K. thanks the EU Innovative Training Network (ProSA) for a studentship. N.T. is funded as a Canadian Institutes of Health Research new investigator and a Michael Smith Foundation of Health Research (MSFHR) career investigator. N.T. thanks Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN 418262-12. A.M.B. is funded as a National Health and Medical Research Senior Research Fellow (1022688). This work was supported by the Victorian Life Sciences Computation Initiative, an initiative of the Victorian Government, Australia.

Author information

Authors and Affiliations

Authors

Contributions

E.C., M.K., G.J.C., P.D.C., B.T.P., E.K.L. and L.A.-J. performed experiments and analyzed results; A.M.B. and M.W. analyzed results; F.H. conceived the project, designed experiments and analyzed results; N.T. conceived the project, designed experiments, analyzed results and wrote the manuscript; and C.J.J. conceived the project, designed experiments, performed experiments, analyzed results and wrote the manuscript.

Corresponding authors

Correspondence to Nobuhiko Tokuriki or Colin J Jackson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–3. (PDF 1751 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Campbell, E., Kaltenbach, M., Correy, G. et al. The role of protein dynamics in the evolution of new enzyme function. Nat Chem Biol 12, 944–950 (2016). https://doi.org/10.1038/nchembio.2175

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2175

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing