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Structure and dynamics of a primordial catalytic fold generated by in vitro evolution

Nature Chemical Biology volume 9, pages 81–83 (2013)Cite this article

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Abstract

Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

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Figure 1: Changes in primary sequence and 3D structure upon directed evolution of the hRXRα scaffold to the ligase enzyme 10C.
Figure 2: Conformational dynamics of ligase enzyme 10C.
Figure 3: Substrate-binding surface of ligase 10C probed by NMR and alanine scanning.

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Biological Magnetic Resonance Data Bank

Protein Data Bank

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Protein Data Bank

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Acknowledgements

We thank M. Golynskiy and A. Pohorille for helpful discussions; Z. Sachs, F.P. Seebeck, J.W. Szostak and F. Hollfelder for comments on the manuscript; and R. Majerle for isothermal titration calorimetry instrument use. This work was supported by the US National Aeronautics and Space Administration (NASA) Agreement no. NNX09AH70A through the NASA Astrobiology Institute–Ames Research Center (to F.-A.C., A.M., L.C. and B.S.); the Minnesota Medical Foundation (to B.S.) and the US National Institutes of Health (NIH) (T32 GM08347 to J.C.H., T32 DE007288 to L.R.M., GM100310 to G.V. and P41 RR001209). Stanford Synchrotron Radiation Lightsource (SSRL) operations are funded by the US Department of Energy (DOE)–Basic Energy Sciences. The SSRL Structural Molecular Biology program is supported by NIH–National Center for Research Resources and DOE–Biological Environmental Resarch.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

    Fa-An Chao, Aleardo Morelli, John C Haugner III, Lewis Churchfield, Leonardo N Hagmann, Larry R Masterson, Gianluigi Veglia & Burckhard Seelig

  2. BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA

    Aleardo Morelli, John C Haugner III, Lewis Churchfield, Leonardo N Hagmann & Burckhard Seelig

  3. Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA

    Lei Shi & Gianluigi Veglia

  4. SLAC National Accelerator Laboratory, Stanford Synchrotron Radiation Lightsource, Menlo Park, California, USA

    Ritimukta Sarangi

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  1. Fa-An Chao
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Contributions

G.V. and B.S. designed the project; A.M., J.C.H., L.C. and L.N.H. expressed and assayed proteins; F.-A.C. carried out all NMR and isothermal titration calorimetry experiments; F.-A.C. and L.S. calculated the structure; R.S. performed the EXAFS measurements, all authors analyzed the data; and F.-A.C., L.R.M., G.V. and B.S. wrote the paper.

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Correspondence to Burckhard Seelig.

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The authors declare no competing financial interests.

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Chao, FA., Morelli, A., III, J. et al. Structure and dynamics of a primordial catalytic fold generated by in vitro evolution. Nat Chem Biol 9, 81–83 (2013). https://doi.org/10.1038/nchembio.1138

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