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. 2012 Jan 10;2(12):1006–1013. doi: 10.1007/s13238-011-1121-3

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

Cheng Zhu 1, Changsheng Zhang 1, Huanhuan Liang 1, Luhua Lai 1,2,✉
PMCID: PMC4875247  PMID: 22231358

Abstract

Functional proteins designed de novo have potential application in chemical engineering, agriculture and healthcare. Metal binding sites are commonly used to incorporate functions. Based on a de novo designed protein DS119 with a βαβ structure, we have computationally engineered zinc binding sites into it using a home-made searching program. Seven out of the eight designed sequences tested were shown to bind Zn2+ with micromolar affinity, and one of them bound Zn2+ with 1:1 stoichiometry. This is the first time that metalloproteins with an α, β mixed structure have been designed from scratch.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-011-1121-3 and is accessible for authorized users.

Keywords: βαβ, de novo, design, folding, zincbinding

Electronic supplementary material

13238_2011_1121_MOESM1_ESM.pdf (909.2KB, pdf)

Supplementary material, approximately 909 KB.

Footnotes

These authors contributed equally to the work.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-011-1121-3 and is accessible for authorized users.

References

  1. Ambroggio X.I., Kuhlman B. Computational design of a single amino acid sequence that can switch between two distinct protein folds. J Am Chem Soc. 2006;128:1154–1161. doi: 10.1021/ja054718w. [DOI] [PubMed] [Google Scholar]
  2. Berg J.M., Shi Y.G. The galvanization of biology: a growing appreciation for the roles of zinc. Science. 1996;271:1081–1085. doi: 10.1126/science.271.5252.1081. [DOI] [PubMed] [Google Scholar]
  3. Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. The Protein Data Bank. Nucleic Acids Res. 2000;28:235–242. doi: 10.1093/nar/28.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cerasoli E., Sharpe B.K., Woolfson D.N. ZiCo: a peptide designed to switch folded state upon binding zinc. J Am Chem Soc. 2005;127:15008–15009. doi: 10.1021/ja0543604. [DOI] [PubMed] [Google Scholar]
  5. Choma C.T., Lear J.D., Nelson M.J., Dutton P.L., Robertson D.E., Degrado W.F. DESIGN OF A HEME-BINDING 4-HELIX BUNDLE. J Am Chem Soc. 1994;116:856–865. doi: 10.1021/ja00082a005. [DOI] [Google Scholar]
  6. Christianson D.W., Fierke C.A. Carbonic anhydrase: Evolution of the zinc binding site by nature and by design. Acc Chem Res. 1996;29:331–339. doi: 10.1021/ar9501232. [DOI] [Google Scholar]
  7. Clarke N.D., Yuan S.M. Metal search: a computer program that helps design tetrahedral metal-binding sites. Proteins. 1995;23:256–263. doi: 10.1002/prot.340230214. [DOI] [PubMed] [Google Scholar]
  8. De Maeyer M., Desmet J., Lasters I. All in one: a highly detailed rotamer library improves both accuracy and speed in the modelling of sidechains by dead-end elimination. Fold Des. 1997;2:53–66. doi: 10.1016/S1359-0278(97)00006-0. [DOI] [PubMed] [Google Scholar]
  9. Dwyer M.A., Looger L.L., Hellinga H.W. Computational design of a Zn2 + receptor that controls bacterial gene expression. Proc Natl Acad Sci U S A. 2003;100:11255–11260. doi: 10.1073/pnas.2032284100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dyer R.B., Gai F., Woodruff W.H. Infrared studies of fast events in protein folding. Acc Chem Res. 1998;31:709–716. doi: 10.1021/ar970343a. [DOI] [Google Scholar]
  11. Fry H.C., Lehmann A., Saven J.G., DeGrado W.F., Therien M. J. Computational design and elaboration of a de novo heterotetrameric alpha-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore. J Am Chem Soc. 2010;132:3997–4005. doi: 10.1021/ja907407m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Handel T.M., Williams S.A., DeGrado W.F. Metal ion-dependent modulation of the dynamics of a designed protein. Science. 1993;261:879–885. doi: 10.1126/science.8346440. [DOI] [PubMed] [Google Scholar]
  13. Hellinga H.W., Richards F.M. Construction of new ligand binding sites in proteins of known structure. I. Computer-aided modeling of sites with pre-defined geometry. J Mol Biol. 1991;222:763–785. doi: 10.1016/0022-2836(91)90510-D. [DOI] [PubMed] [Google Scholar]
  14. Holm R.H., Kennepohl P., Solomon E.I. Structural and functional aspects of metal sites in biology. Chem Rev. 1996;96:2239–2314. doi: 10.1021/cr9500390. [DOI] [PubMed] [Google Scholar]
  15. Kaplan J., DeGrado W.F. De novo design of catalytic proteins. Proc Natl Acad Sci U S A. 2004;101:11566–11570. doi: 10.1073/pnas.0404387101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kiyokawa T., Kanaori K., Tajima K., Koike M., Mizuno T., Oku J.I., Tanaka T. Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded alpha-helical coiled-coil toward a prototype for a metalloenzyme. J Pept Res. 2004;63:347–353. doi: 10.1111/j.1399-3011.2004.00109.x. [DOI] [PubMed] [Google Scholar]
  17. Li W.F., Zhang J., Wang W. Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations. Proteins. 2007;67:338–349. doi: 10.1002/prot.21312. [DOI] [PubMed] [Google Scholar]
  18. Li W.F., Zhang J., Wang J., Wang W. Metal-coupled folding of Cys2His2 zinc-finger. J Am Chem Soc. 2008;130:892–900. doi: 10.1021/ja075302g. [DOI] [PubMed] [Google Scholar]
  19. Liang H.H., Chen H., Fan K.Q., Wei P., Guo X.R., Jin C.W., Zeng C., Tang C., Lai L.H. De novo design of a beta alpha beta motif. Angew Chem Int Ed Engl. 2009;48:3301–3303. doi: 10.1002/anie.200805476. [DOI] [PubMed] [Google Scholar]
  20. Lombardi A., Summa C.M., Geremia S., Randaccio L., Pavone V., DeGrado W.F. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Proc Natl Acad Sci U S A. 2000;97:6298–6305. doi: 10.1073/pnas.97.12.6298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lu Y., Berry S.M., Pfister T.D. Engineering novel metalloproteins: design of metal-binding sites into native protein scaffolds. Chem Rev. 2001;101:3047–3080. doi: 10.1021/cr0000574. [DOI] [PubMed] [Google Scholar]
  22. Lu Y., Yeung N., Sieracki N., Marshall N.M. Design of functional metalloproteins. Nature. 2009;460:855–862. doi: 10.1038/nature08304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matzapetakis M., Farrer B.T., Weng T.C., Hemmingsen L., Penner-Hahn J.E., Pecoraro V.L. Comparison of the binding of cadmium(II), mercury(II), and arsenic(III) to the de novo designed peptides TRI L12C and TRI L16C. J Am Chem Soc. 2002;124:8042–8054. doi: 10.1021/ja017520u. [DOI] [PubMed] [Google Scholar]
  24. Meinnel T., Blanquet S., Dardel F. A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase. J Mol Biol. 1996;262:375–386. doi: 10.1006/jmbi.1996.0521. [DOI] [PubMed] [Google Scholar]
  25. Miller J.C., Holmes M.C., Wang J.B., Guschin D.Y., Lee Y.L., Rupniewski I., Beausejour C.M., Waite A.J., Wang N.S., Kim K. A., et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007;25:778–785. doi: 10.1038/nbt1319. [DOI] [PubMed] [Google Scholar]
  26. Müller H.N., Skerra A. Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification. Biochemistry. 1994;33:14126–14135. doi: 10.1021/bi00251a023. [DOI] [PubMed] [Google Scholar]
  27. Nomura A., Sugiura Y. Hydrolytic reaction by zinc finger mutant peptides: successful redesign of structural zinc sites into catalytic zinc sites. Inorg Chem. 2004;43:1708–1713. doi: 10.1021/ic034931y. [DOI] [PubMed] [Google Scholar]
  28. Pasquinelli R.S., Shepherd R.E., Koepsel R.R., Zhao A., Ataai M.M. Design of affinity tags for one-step protein purification from immobilized zinc columns. Biotechnol Prog. 2000;16:86–91. doi: 10.1021/bp990139h. [DOI] [PubMed] [Google Scholar]
  29. Petros A.K., Reddi A.R., Kennedy M.L., Hyslop A.G., Gibney B.R. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites. Inorg Chem. 2006;45:9941–9958. doi: 10.1021/ic052190q. [DOI] [PubMed] [Google Scholar]
  30. Phillips N.B., Wan Z.L., Whittaker L., Hu S.Q., Huang K., Hua Q. X., Whittaker J., Ismail-Beigi F., Weiss M.A. Supramolecular protein engineering: design of zinc-stapled insulin hexamers as a long acting depot. J Biol Chem. 2010;285:11755–11759. doi: 10.1074/jbc.C110.105825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Proudfoot C., McPherson A.L., Kolb A.F., Stark W.M. Zinc finger recombinases with adaptable DNA sequence specificity. PLoS One. 2011;6:e19537. doi: 10.1371/journal.pone.0019537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reddi A.R., Guzman T.R., Breece R.M., Tiemey D.L., Gibney B.R. Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed Metallopeptides. J Am Chem Soc. 2007;129:12815–12827. doi: 10.1021/ja073902+. [DOI] [PubMed] [Google Scholar]
  33. Regan L., Clarke N.D. A tetrahedral zinc(II)-binding site introduced into a designed protein. Biochemistry. 1990;29:10878–10883. doi: 10.1021/bi00501a003. [DOI] [PubMed] [Google Scholar]
  34. Shults M.D., Pearce D.A., Imperiali B. Modular and tunable chemosensor scaffold for divalent zinc. J Am Chem Soc. 2003;125:10591–10597. doi: 10.1021/ja0355980. [DOI] [PubMed] [Google Scholar]
  35. Smith B.A., Hecht M.H. Novel proteins: from fold to function. Curr Opin Chem Biol. 2011;15:421–426. doi: 10.1016/j.cbpa.2011.03.006. [DOI] [PubMed] [Google Scholar]
  36. Stevens F.J. Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association. Biochemistry. 1986;25:981–993. doi: 10.1021/bi00353a006. [DOI] [PubMed] [Google Scholar]
  37. Wade W.S., Koh J.S., Han N., Hoekstra D.M., Lerner R.A. Engineering Metal Coordination Sites into the Antibody Light-Chain. J Am Chem Soc. 1993;115:4449–4456. doi: 10.1021/ja00064a005. [DOI] [Google Scholar]
  38. Winzor D.J. Analytical exclusion chromatography. J Biochem Biophys Methods. 2003;56:15–52. doi: 10.1016/S0165-022X(03)00071-X. [DOI] [PubMed] [Google Scholar]
  39. Wu J., Kandavelou K., Chandrasegaran S. Custom-designed zinc finger nucleases: what is next? Cell Mol Life Sci. 2007;64:2933–2944. doi: 10.1007/s00018-007-7206-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

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13238_2011_1121_MOESM1_ESM.pdf (909.2KB, pdf)

Supplementary material, approximately 909 KB.

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