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A protein engineered to bind uranyl selectively and with femtomolar affinity

Abstract

Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM); however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30–60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

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Figure 1: Uranyl sequestration strategy.
Figure 2: The main steps in computational screening and design of uranyl-binding proteins.
Figure 3: Uranyl-binding affinity and selectivity of SUP.
Figure 4: Uranyl–SUP crystal structure.
Figure 5: Immobilized SUP provides a useful manifold for a variety of applications.

References

  1. Nozaki, Y. A fresh look at element distribution in the North Pacific. Eos Trans. Am. Geophys. Union 78, 221–227 (1997).

    Google Scholar 

  2. Tabushi, I., Kobuke, Y. & Nishiya, T. Extraction of uranium from seawater by polymer-bound macrocyclic hexaketone. Nature 280, 665–666 (1979).

    Article  CAS  Google Scholar 

  3. Kanno, M. Present status of study on extraction of uranium from sea water. J. Nucl. Sci. Technol. 21, 1–9 (1984).

    Article  CAS  Google Scholar 

  4. Egawa, H., Nonaka, T. & Nakayama, M. Recovery of uranium from seawater. 7. Concentration and separation of uranium in acidic eluate. Ind. Eng. Chem. Res. 29, 2273–2277 (1990).

    Article  CAS  Google Scholar 

  5. Sather, A. C., Berryman, O. B., Ajami, D. & Rebek, J. Jr Selective recognition and extraction of the uranyl ion. J. Am. Chem. Soc. 132, 13572–13574 (2010).

    Article  CAS  Google Scholar 

  6. Franczyk, T. S., Czerwinski, K. R. & Raymond, K. N. Stereognostic coordination chemistry. 1. The design and synthesis of chelators for the uranyl ion. J. Am. Chem. Soc. 114, 8138–8146 (1992).

    Article  CAS  Google Scholar 

  7. Gordon, A. E., Xu, J., Raymond, K. N. & Durbin, P. Rational design of sequestering agents for plutonium and other actinides. Chem. Rev. 103, 4207–4282 (2003).

    Article  Google Scholar 

  8. Gong, H. Y., Rambo, B. M., Karnas, E., Lynch, V. M. & Sessler, J. L. A ‘Texas-sized’ molecular box that forms an anion-induced supramolecular necklace. Nature Chem. 2, 406–409 (2010).

    Article  CAS  Google Scholar 

  9. Wegner, S. V., Boyaci, H., Chen, H., Jensen, M. P. & He, C. Engineering a uranyl-specific binding protein from NikR. Angew. Chem. Int. Ed. 48, 2339–2341 (2009).

    Article  CAS  Google Scholar 

  10. Lee, J. H., Wang, Z., Liu, J. & Lu, Y. Highly sensitive and selective colorimetric sensors for uranyl (UO22+): development and comparison of labeled and label-free DNAzyme–gold nanoparticle systems. J. Am. Chem. Soc. 130, 14217–14226 (2008).

    Article  CAS  Google Scholar 

  11. LeClainche, L. & Vita, C. Selective binding of uranyl cation by a novel calmodulin peptide. Environ. Chem. Lett. 4, 45–49 (2006).

    Article  CAS  Google Scholar 

  12. Manos M. J. & Kanatzidis M. G. Layered metal sulfides capture uranium from seawater. J. Am. Chem. Soc., 134 (39), 16441–16446 (2012).

    Article  CAS  Google Scholar 

  13. Carboni, M., Abney, C. W., Liu, S. & Lin, W. Highly porous and stable metal–organic frameworks for uranium extraction. Chem. Sci. 4, 2396–2402 (2013).

    Article  CAS  Google Scholar 

  14. Changlea, A. et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387 (2003).

    Article  Google Scholar 

  15. Aisen, P., Leibman, A. & Zweier, J. Stoichiometric and site characteristics of the binding of iron to human transferrin. J. Biol. Chem. 253, 1930–1937 (1978).

    CAS  PubMed  Google Scholar 

  16. Banci, L. et al. Affinity gradients drive copper to cellular destinations. Nature 465, 367–368 (2010).

    Article  Google Scholar 

  17. Georgiou, G. et al. Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp′–OmpA′–beta-lactamase fusions. Protein Eng. 9, 239–247 (1996).

    Article  CAS  Google Scholar 

  18. Zhang, C. & Lai, L. Automatch: target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds. Proteins 80, 1078–1094 (2012).

    Article  CAS  Google Scholar 

  19. Pible, O., Guilbaud, P., Pellequer, J. L., Vidaud, C. & Quéméneur, E. Biochimie 88, 1631–1638 (2006).

    Article  CAS  Google Scholar 

  20. Zeikus, J. G. & Wolee, R. S. Methanobacterium thermoautotrophicus sp. n., an anaerobic, autotrophic, extreme thermophile. J. Bacteriol. 109, 707–715 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Saito, K. & Miyauchi, T. Chemical forms of uranium in artificial seawater. J. Nucl. Sci. Technol. 19, 145–150 (1982).

    Article  CAS  Google Scholar 

  22. Regan, L. & DeGrado, W. F. Characterization of a helical protein designed from first principles. Science 241, 976–978 (1988).

    Article  CAS  Google Scholar 

  23. Lovejoy, B. et al. Crystal structure of a synthetic triple-stranded alpha-helical bundle. Science 259, 1288–1293 (1993).

    Article  CAS  Google Scholar 

  24. Wendt, H., Berger, C., Baici, A., Thomas, R. M. & Bosshard, H. R. Kinetics of folding of leucine zipper domains. Biochemistry 34, 4097–4107 (1995).

    Article  CAS  Google Scholar 

  25. Pordea, A. & Ward, T. R Artificial metalloenzymes: combining the best features of homogenous and enzymatic catalysis. Synlett 20, 3225–3236 (2009).

    Google Scholar 

  26. DeGrado, W. F., Summa, C. M., Pavone, V., Nastri, F. & Lombardi, A. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68, 779–819 (1999).

    Article  CAS  Google Scholar 

  27. Dutton, P. L. & Moser, C. C. Engineering enzymes. Faraday Discuss. 148, 443–448 (2011).

    Article  CAS  Google Scholar 

  28. Khare, S. D. et al. Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nature Chem. Biol. 8, 294–300 (2012).

    Article  CAS  Google Scholar 

  29. Azoitei, M. L. et al. Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334, 373–376 (2011).

    Article  CAS  Google Scholar 

  30. Radford, R. J., Brodin, J. D., Salgado, E. N. & Tezcan, A. Expanding the utility of proteins as platforms for coordination chemistry. Coord. Chem. Rev. 225, 790–803 (2011).

    Article  Google Scholar 

  31. Brodin, J. D. et al. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nature Chem. 4, 375–382 (2012).

    Article  CAS  Google Scholar 

  32. Saven, J. G. Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins. Curr. Opin. Chem. Biol. 15, 452–457 (2011).

    Article  CAS  Google Scholar 

  33. Privett, H. K. et al. Iterative approach to computational enzyme design. Proc. Natl Acad. Sci. USA 109, 3790–3795 (2012).

    Article  CAS  Google Scholar 

  34. Liu, S. et al. Nonnatural protein–protein interaction-pair design by key residues grafting. Proc. Natl Acad. Sci. USA 104, 5330–5335 (2007).

    Article  CAS  Google Scholar 

  35. Fleishman, S. J. et al. Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332, 816–821 (2011).

    Article  CAS  Google Scholar 

  36. Lu, Y., Yeung, N., Sieracki, N. & Marshall, N. M. Design of functional metalloproteins. Nature 460, 855–862 (2009).

    Article  CAS  Google Scholar 

  37. Touw, D. S., Nordman, C. E., Stuckey, J. A. & Pecoraro, V. L. Identifying important structural characteristics of arsenic resistance proteins by using designed three stranded coils. Proc. Natl Acad. Sci. USA 104, 11969–11974 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy, under contract number DE-FG02-07ER15865 to C.H., and at Argonne National Laboratory (M.J.) under contract number DE-AC02-06CH11357, the Dreyfus Foundation Postdoctoral Program in Environmental Chemistry to S.O., the Ministry of Science and Technology of China (2009CB918500) and the National Natural Science Foundation of China (21173013, 11021463) to L.L. Use of the Advanced Photon Source for protein crystallography data collection at beamlines LS/CA-CAT (21-ID-F) and NE-CAT (24-ID-C) was supported by the Office of Basic Energy Sciences of the US Department of Energy under contract number DE-AC02-06CH11357. We thank S. F. Reichard for editing the manuscript and C. Yang and L. Lan for experimental support.

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Contributions

C.H. conceived the project. C.H. and L.L. designed the experiment. C.Z. and L.L. performed the initial screening. W.Z. and C.J.L. performed subcloning of virtual hits. S.O. and C.J.L. expressed, purified and tested all first-generation virtual hits. L.Z., S.O. and M.B. designed second-generation mutants. L.Z., M.B. and J.L. expressed and tested all protein derivatives and designed all later-generation mutants. L.Z., M.B., M.P.J. and C.H. analysed the data and M.B. and L.Z. co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Luhua Lai or Chuan He.

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The authors declare no competing financial interests.

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Zhou, L., Bosscher, M., Zhang, C. et al. A protein engineered to bind uranyl selectively and with femtomolar affinity. Nature Chem 6, 236–241 (2014). https://doi.org/10.1038/nchem.1856

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