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.

  • Article
  • Published:

An efficient, step-economical strategy for the design of functional metalloproteins

Nature Chemistry volume 11, pages 434–441 (2019)Cite this article

Subjects

Abstract

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein–protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres—including a penta-His-coordinated non-haem Fe site—and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Design strategies for the construction of functional metalloproteins.
Fig. 2: Implementation of MASCoT.
Fig. 3: Structural, spectroscopic and analytical data on Cu-, Fe-, and VIVO bound metalloproteins.
Fig. 4: Mn binding by CH2E and CH2EY.
Fig. 5: Nitric oxide binding properties of Fe–CH3Y*.

Similar content being viewed by others

Data availability

The principal data supporting the findings of this work are available within the figures and the Supplementary Information. Coordinates and structure factor files for Co–H3 (6DYI), apo–CH3 (6DYB), Co–CH3 (6DYC), Fe–CH3 (6DYE), Cu–CH3 (6DYD), Fe–CH3Y (6DYG), Cu–CH3Y (6DYF), VIVO–CH3Y (6DYH), Mn–CH2E (6DY6), Fe–CH2E (6DY4), Mn–CH2EY (6DY8), Fe–CH3Y* (6DYJ), FeNO–CH3Y* (6DYK), and VIVO–CH3Y* (6DYL) have been deposited to the Protein Data Bank with the corresponding PDB ID codes. Additional data that support the findings of this study are available from the corresponding author on request.

References

  1. Gray, H. B., Stiefel, E. I., Valentine, J. S. & Bertini, I. Biological Inorganic Chemistry: Structure and Reactivity (University Science Books, Sausalito, 2007).

  2. Chen, A. Y. et al. Targetting metalloenzymes for therapeutic intervention. Chem. Rev. 119, 1323–1455 (2019).

    Article  CAS  Google Scholar 

  3. Matés, J. M., Segura, J. A., Alonso, F. J. & Márques, J. Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch. Toxicol. 82, 273–299 (2008).

    Article  Google Scholar 

  4. Corbin, B. D. et al. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science 319, 962–965 (2008).

    Article  CAS  Google Scholar 

  5. Hayden, J. A., Brophy, M. B., Cunden, L. S. & Nolan, E. M. High-affinity manganese coordination by human calprotectin is calcium-dependent and requires a histidine-rich site formed at the dimer interface. J. Am. Chem. Soc. 135, 775–787 (2012).

    Article  Google Scholar 

  6. Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 angstrom. Nature 473, 55–65 (2011).

    Article  CAS  Google Scholar 

  7. de Montellano, P. R. O. Cytochrome P450: Structure, Mechanism, and Biochemistry (Kluwer Academic/Plenum, New York, 2005).

  8. Yu, F. et al. Protein design: toward functional metalloenzymes. Chem. Rev. 114, 3495–3578 (2014).

    Article  CAS  Google Scholar 

  9. Schwizer, F. et al. Artificial metalloenzymes: reaction scope and optimization strategies. Chem. Rev. 118, 142–231 (2018).

    Article  CAS  Google Scholar 

  10. Lu, Y., Yeung, N., Sieracki, N. & Marshall, N. M. Design of functional metalloproteins. Nature 460, 855–562 (2009).

    Article  CAS  Google Scholar 

  11. Soler-Illia, G. J. A. A., Sanchez, C., Lebeau, B. & Patarin, J. Chemical strategies to design textured materials: from microporous and mesoporous oxides to nanonetworks and hierarchical structures. Chem. Rev. 102, 4093–4138 (2002).

    Article  Google Scholar 

  12. Fürstner, A. Olefin metathesis and beyond. Angew. Chem. Int. Ed. 39, 3012–3043 (2000).

    Article  Google Scholar 

  13. Wender, P. A., Verma, V. A., Paxton, T. J. & Pillow, T. H. Function-oriented synthesis, step economy, and drug design. Acc. Chem. Res. 41, 40–49 (2008).

    Article  CAS  Google Scholar 

  14. Hyster, T. K., Knorr, L., Ward, T. R. & Rovis, T. Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C-H activation. Science 338, 500–503 (2012).

    Article  CAS  Google Scholar 

  15. Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing nobel metals in place of iron. Nature. 534, 534–537 (2016).

    Article  CAS  Google Scholar 

  16. Bos, J., Fusetti, F., Diressen, A. J. M. & Roelfes, G. Enantioselective artificial metalloenzymes by creation of a novel active site at the protein dimer interface. Angew. Chem. Int. Ed. 51, 7472–7475 (2012).

    Article  CAS  Google Scholar 

  17. Cavazza, C. et al. Crystallographic snapshots of the reaction of aromatic C–H with O2 catalyzed by a protein-bound iron complex. Nat. Chem. 2, 1069–1076 (2010).

    Article  CAS  Google Scholar 

  18. Hayashi, T., Hilvert, D. & Green, A. P. Engineered metalloenzymes with non-canonical coordination environments. Chem. Eur. J. 24, 1–11 (2018).

    Article  Google Scholar 

  19. Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016).

    Article  CAS  Google Scholar 

  20. Yang, H. et al. Evolving artificial metalloenzymes via random mutagenesis. Nat. Chem. 10, 318–324 (2018).

    Article  CAS  Google Scholar 

  21. Mann, S. I., Heinisch, T., Ward, T. R. & Borovik, A. S. Peroxide activation regulated by hydrogen bonds within artificial Cu proteins. J. Am. Chem. Soc. 139, 17289–17292 (2017).

    Article  CAS  Google Scholar 

  22. Yeung, N. et al. Rational design of a structural and functional nitric oxide reductase. Nature 462, 1079–1082 (2009).

    Article  CAS  Google Scholar 

  23. Toscano, M. D., Woycechowsky, K. J. & Hilvert, D. Minimalist active-site redesign: teaching old enzymes new tricks. Angew. Chem. Int. Ed. 46, 3212–3236 (2007).

    Article  CAS  Google Scholar 

  24. Lombardi, A. et al. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Proc. Natl Acad. Sci. USA 97, 6298–6305 (2000).

    Article  CAS  Google Scholar 

  25. Reig, A. J. et al. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins. Nat. Chem. 4, 900–906 (2012).

    Article  CAS  Google Scholar 

  26. Koder, R. L. et al. Design and engineering of an O2 transport protein. Nature 458, 305–310 (2009).

    Article  CAS  Google Scholar 

  27. Lombardi, A. et al. Miniaturized metalloproteins: applications to iron-sulfur proteins. Proc. Natl Acad. Sci. USA 97, 11922–11927 (2000).

    Article  CAS  Google Scholar 

  28. Zastrow, M. L., Peacock, A. F. A., Stuckey, J. A. & Pecoraro, V. L. Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nat. Chem. 4, 118–123 (2012).

    Article  CAS  Google Scholar 

  29. Der, B. S., Edwards, D. R. & Kuhlman, B. Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering. Biochemistry 51, 3933–3940 (2012).

    Article  CAS  Google Scholar 

  30. Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew. Chem. Int. Ed. 57, 4143–4148 (2018).

    Article  CAS  Google Scholar 

  31. Reetz, M. T. Combinatorial and evolution-based methods in the creation of enantioselective catalysts. Angew. Chem. Int. Ed. 40, 284–310 (2001).

    Article  CAS  Google Scholar 

  32. Rufo, C. M. et al. Short peptides self-assemble to produce catalytic amyloids. Nat. Chem. 6, 303–309 (2014).

    Article  CAS  Google Scholar 

  33. Salgado, E. N., Radford, R. J. & Tezcan, F. A. Metal-directed protein self-assembly. Acc. Chem. Res. 43, 661–672 (2010).

    Article  CAS  Google Scholar 

  34. Churchfield, L. A., Medina-Morales, A., Brodin, J. D., Perez, A. & Tezcan, F. A. De novo design of an allosteric metalloprotein assembly with strained disulfide bonds. J. Am. Chem. Soc. 138, 13163–13166 (2016).

    Article  CAS  Google Scholar 

  35. Churchfield, L. A., Alberstein, R. G., Williamson, L. M. & Tezcan, F. A. Determining the structural and energetic basis of allostery in a de novo designed metalloprotein assembly. J. Am. Chem. Soc. 140, 10043–10053 (2018).

    Article  CAS  Google Scholar 

  36. Song, W. J. & Tezcan, F. A. A designed supramolecular protein assembly with in vivo enzymatic activity. Science 346, 1525–1528 (2014).

    Article  CAS  Google Scholar 

  37. Song, W. J., Yu, J. & Tezcan, F. A. Importance of scaffold flexibility/rigidity in the design and directed evolution of artificial metallo-β-lactamases. J. Am. Chem. Soc. 139, 16772–16779 (2017).

    Article  CAS  Google Scholar 

  38. Eijsink, V. G. H. et al. Rational engineering of enzyme stability. J. Biotech. 113, 105–120 (2004).

    Article  CAS  Google Scholar 

  39. Dombkowski, A. A., Sultana, K. Z. & Craig, D. B. Protein disulfide engineering. FEBS Lett. 588, 206–212 (2014).

    Article  CAS  Google Scholar 

  40. Robertson, D. E. et al. Design and synthesis of multi-haem proteins. Nature 368, 425–432 (1994).

    Article  CAS  Google Scholar 

  41. Lichtenstein, B. R. et al. eE Engineering oxidoreductases: maquette proteins designed from scratch. Biochem. Soc. Trans. 40, 561–566 (2012).

    Article  CAS  Google Scholar 

  42. Gibney, B. R. et al. Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette. Biochemistry 39, 11041–11049 (2000).

    Article  CAS  Google Scholar 

  43. Faraone-Mennella, J., Tezcan, F. A., Gray, H. B. & Winkler, J. R. Stability and folding kinetics of structurally characterized cytochrome c-b 562. Biochemistry 45, 10504–10511 (2006).

    Article  CAS  Google Scholar 

  44. Black, D. S. C. & Hartshorn, A. J. Ligand design and synthesis. Coord. Chem. Rev. 9, 219–274 (1972).

    Article  Google Scholar 

  45. Goncharenko, K. V., Vit, A., Blankenfeldt, W. & Seebeck, F. P. Structure of the sulfoxide synthase EgtB from the ergothioneine biosynthetic pathway. Ang. Chem. Int. Ed. 54, 2821–2824 (2015).

    Article  CAS  Google Scholar 

  46. Krissinel, E. & Henrick, K. Interface of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  47. Addison, A. W., Rao, T. N., Reedijk, J., van Rijn, J. & Verschoor, G. C. Synthesis, structure, and spectroscopic properties of copper(II) compounds containing nitrogen–sulphur donor ligands; the crystal and molecular structure of aqua[1,7-bis(N-methylbenzimidazol-2’-yl)-2,6-dithiaheptane]copper(II) perchlorate. J. Chem. Soc. Dalton. Trans. 1984, 1349–1356 (1984).

  48. Smith, T. S., LoBrutto, R. & Pecoraro, V. L. Paramagnetic spectroscopy of vanadyl complexes and its applications to biological systems. Coord. Chem. Rev. 228, 1–18 (2002).

    Article  CAS  Google Scholar 

  49. Shi, R. et al. Crystal structures of apo and metal-bound forms of the UreE protein from Helicobacter pylori: role of multiple metal binding sites. Biochemistry 49, 7080–7088 (2010).

    Article  CAS  Google Scholar 

  50. Zhu, G., Koszelak-Rosenblum, M., Connelly, S. M., Dumont, M. E. & Malkowski, M. G. The crystal structure of an integral membrane fatty acid α-hydroxylase. J. Biol. Chem. 290, 29820–29833 (2015).

    Article  CAS  Google Scholar 

  51. Parmar, A. S., Pike, D. & Nanda, V. in Protein Design Methods and Applications (ed. Köhler, V.) Ch. 12 (Humana, New York, 2014).

  52. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A New generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  PubMed  Google Scholar 

  53. Zastrow, M. L. & Pecoraro, V. L. Designing hydrolytic zinc metalloenzymes. Biochemistry 53, 957–978 (2014).

    Article  CAS  Google Scholar 

  54. Vita, C., Roumestand, C., Toma, F. & Ménez, A. Scorpion toxins as natural scaffolds for protein engineering. Proc. Natl Acad. Sci. USA 92, 6404–6408 (1995).

    Article  CAS  Google Scholar 

  55. Brodin, J. D. et al. Evolution of metal selectivity in templated protein interfaces. J. Am. Chem. Soc. 132, 8610–8617 (2010).

    Article  CAS  Google Scholar 

  56. Hosseinzadeh, P. et al. Enhancing Mn(II)-binding and manganese peroxidase activity in a designed cytochrome c peroxidase through fine-tuning secondary-sphere interactions. Biochemistry 55, 1494–1502 (2016).

    Article  CAS  Google Scholar 

  57. Golynskiy, M. V., Gunderson, W. A., Hendrich, M. P. & Cohen, S. M. Metal binding studies and EPR spectroscopy of the manganese transport regulator MntR. Biochemistry 45, 15359–15372 (2006).

    Article  CAS  Google Scholar 

  58. Cotruvo, J. A. & Stubbe, J. Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study. Metallomics. 4, 1020–1036 (2012).

    Article  CAS  Google Scholar 

  59. Koepke, J. et al. pH modulates the quinone position in the photosynthetic reaction center from Rhodobacter sphaeroides in the neutral and charge separated states. J. Mol. Biol. 371, 396–409 (2007).

    Article  CAS  Google Scholar 

  60. Chu, R. et al. Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography. J. Mol. Biol. 323, 253–262 (2002).

    Article  CAS  Google Scholar 

  61. Pasternak, A., Kaplan, J., Lear, J. D. & DeGrado, W. F. Proton and metal ion-dependent assembly of a model diiron protein. Protein Sci. 10, 958–969 (2001).

    Article  CAS  Google Scholar 

  62. Zhuang, J. et al. Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin. Inorg. Chem. 43, 8218–8220 (2004).

    Article  CAS  Google Scholar 

  63. McLaughlin, M. P. et al. Azurin as a protein scaffold for a low-coordinate non-heme iron site with a small-molecule binding pocket. J. Am. Chem. Soc. 134, 19746–19757 (2012).

    Article  CAS  Google Scholar 

  64. Chakraborty, S. et al. Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase. Angew. Chem. Int. Ed. 126, 2449–2453 (2014).

    Article  Google Scholar 

  65. Orville, A. M. & Lipscomb, J. D. Simultaneous binding of nitric oxide and isotopically-labeled substrates of inhibitors by reduced protocatechuate 3,4-dioxygenase. J. Biol. Chem. 268, 8596–8607 (1993).

    CAS  PubMed  Google Scholar 

  66. Martini, R. J. et al. Experimental correlation of substrate position with reaction outcome in the aliphatic halogenase, SyrB2. J. Am. Chem. Soc. 137, 6912–6919 (2015).

    Article  Google Scholar 

  67. Li, M. et al. Tuning the electronic structure of octahedral iron complexes [FeL(X)] (L = 1-alkyl-4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane, X = Cl, CH3O, CN, NO). The S = 1/2, S = 3/2 spin equilibrium of [FeLPr(NO)]. Inorg. Chem. 41, 3444–3456 (2002).

    Article  CAS  Google Scholar 

  68. Münck, E. in Physical Methods in Bioinorganic Chemistry (ed. Que, L. Jr) Ch. 6 (University Science Books, Sausalito, 2000).

  69. Ray, M. et al. Structure and magnetic properties of trigonal bipyramidal iron nitrosyl complexes. Inorg. Chem. 38, 3110–3115 (1999).

    Article  CAS  Google Scholar 

  70. Speelman, A. L. et al. Non-heme high-spin {FeNO}6-8 complexes: one ligand platform can do it all. J. Am. Chem. Soc. 140, 11341–11359 (2018).

    Article  CAS  Google Scholar 

  71. Serres, R. G. et al. Structural, spectroscopic, and computational study of an octahedral, non-haem {Fe-NO}6-8 Series: [Fe(NO)(cyclam-ac)]2+/+/0. J. Am. Chem. Soc. 126, 5138–5153 (2004).

    Article  CAS  Google Scholar 

  72. Mbughuni, M. M. et al. Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme. Proc. Natl Acad. Sci. USA 107, 16788–16793 (2010).

    Article  CAS  Google Scholar 

  73. Nakashige, T. G. & Nolan, E. M. Human calprotectin affects the redox speciation of iron. Metallomics 9, 1086–1095 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Science Foundation (grant no. CHE1607145 to F.A.T.). J.R. was supported by a postdoctoral fellowship from the National Institute of General Medical Sciences of the National Institute of Health (grant no. F32GM120981). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource at the Stanford Linear Accelerator Center and the Advanced Light Source at the Lawrence Berkeley National Laboratory, which are supported by the DOE, Office of Science, and Office of Basic Energy Sciences under contracts DE-AC02-76SF00515 and DE-AC02-05CH11231, respectively. We thank C. Moore and M. Gembicky for assistance with XRD experiments.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    Jonathan Rittle & F. Akif Tezcan

  2. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    Mackenzie J. Field & Michael T. Green

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA

    Michael T. Green

  4. Materials Science and Engineering, University of California, San Diego, La Jolla, CA, USA

    F. Akif Tezcan

Authors
  1. Jonathan Rittle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mackenzie J. Field
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael T. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Akif Tezcan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.R. co-conceived the project, designed and performed experiments, analysed data and co-wrote the paper. M.J.F. and M.T.G. performed EPR and Mössbauer experiments. F.A.T. conceived and directed the project and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to F. Akif Tezcan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

The Supplementary Information File that accompanies this manuscript includes detailed experimental protocols and a description of the materials and methods employed. Supplementary tables and figures include data of interest to specialists and that support the arguments found in the main text.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rittle, J., Field, M.J., Green, M.T. et al. An efficient, step-economical strategy for the design of functional metalloproteins. Nat. Chem. 11, 434–441 (2019). https://doi.org/10.1038/s41557-019-0218-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-019-0218-9

This article is cited by

Search

Advanced 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