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.

High-affinity binders selected from designed ankyrin repeat protein libraries

Abstract

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 Å resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Construction of designed AR protein libraries.
Figure 2: Expression, purification and SPR analysis of selected AR proteins.
Figure 3: ELISAs with selected AR proteins.
Figure 4: Crystal structure of the designed AR protein off7 in complex with MBP.
Figure 5: Open sandwich illustrations of the interaction surfaces of AR proteins and their targets.

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. 1

    Groves, M.R. & Barford, D. Topological characteristics of helical repeat proteins. Curr. Opin. Struct. Biol. 9, 383–389 (1999).

    CAS  Article  Google Scholar 

  2. 2

    Andrade, M.A., Perez-Iratxeta, C. & Ponting, C.P. Protein repeats: structures, functions, and evolution. J. Struct. Biol. 134, 117–131 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Kobe, B. & Kajava, A.V. When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends Biochem. Sci. 25, 509–515 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Marcotte, E.M., Pellegrini, M., Yeates, T.O. & Eisenberg, D. A census of protein repeats. J. Mol. Biol. 293, 151–160 (1999).

    CAS  Article  Google Scholar 

  5. 5

    Ellis, J., Dodds, P. & Pryor, T. Structure, function and evolution of plant disease resistance genes. Curr. Opin. Plant Biol. 3, 278–284 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Bork, P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins: Struct. Funct. Genet. 17, 363–374 (1993).

    CAS  Article  Google Scholar 

  7. 7

    Sedgwick, S.G. & Smerdon, S.J. The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biochem. Sci. 24, 311–316 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Suzuki, F. et al. Functional interactions of transcription factor human GA-binding protein subunits. J. Biol. Chem. 273, 29302–29308 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Malek, S., Huxford, T. & Ghosh, G. IκBα functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-κB. J. Biol. Chem. 273, 25427–25435 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Winter, G. & Milstein, C. Man-made antibodies. Nature 349, 293–299 (1991).

    CAS  Article  Google Scholar 

  11. 11

    Plückthun, A. et al. in Antibody Engineering (eds. McCafferty, J., Hoogenboom, H.J. & Chiswell, D.J.) 203–252 (IRL Press, Oxford, 1996).

    Google Scholar 

  12. 12

    Nygren, P.-Å. & Uhlén, M. Scaffolds for engineering novel binding sites in proteins. Curr. Opin. Struct. Biol. 7, 463–469 (1997).

    CAS  Article  Google Scholar 

  13. 13

    Skerra, A. Engineered protein scaffolds for molecular recognition. J. Mol. Recognit. 13, 167–187 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Skerra, A. Imitating the humoral immune response. Curr. Opin. Chem. Biol. 7, 683–693 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Forrer, P., Stumpp, M.T., Binz, H.K. & Plückthun, A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 539, 2–6 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Binz, H.K., Stumpp, M.T., Forrer, P., Amstutz, P. & Plückthun, A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332, 489–503 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Kohl, A. et al. Designed to be stable: crystal structure of a consensus ankyrin repeat protein. Proc. Natl. Acad. Sci. USA 100, 1700–1705 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Mosavi, L.K., Minor, D.L. Jr. & Peng, Z.-y. Consensus-derived structural determinants of the ankyrin repeat motif. Proc. Natl. Acad. Sci. USA 99, 16029–16034 (2002).

    CAS  Article  Google Scholar 

  19. 19

    Main, E.R., Xiong, Y., Cocco, M.J., D'Andrea, L. & Regan, L. Design of stable alpha-helical arrays from an idealized TPR motif. Structure (Camb) 11, 497–508 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Rubin, S.M., Lee, S.Y., Ruiz, E.J., Pines, A. & Wemmer, D.E. Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy. J. Mol. Biol. 322, 425–440 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Hanes, J. & Plückthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Yang, F. et al. Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD. Nat. Struct. Biol. 7, 230–237 (2000).

    CAS  Article  Google Scholar 

  23. 23

    Hon, W.C. et al. Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases. Cell 89, 887–895 (1997).

    CAS  Article  Google Scholar 

  24. 24

    Forrer, P., Tamaskovic, R. & Jaussi, R. Enzyme-linked immunosorbent assay for the measurement of JNK, ERK and p38 kinase activities. Biol. Chem. 379, 1101–1111 (1998).

    CAS  Article  Google Scholar 

  25. 25

    Jones, S. & Thornton, J.M. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Lo Conte, L., Chothia, C. & Janin, J. The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177–2198 (1999).

    CAS  Article  Google Scholar 

  27. 27

    Högbom, M., Eklund, M., Nygren, P.-Å. & Nordlund, P. Structural basis for recognition by an in vitro evolved affibody. Proc. Natl. Acad. Sci. USA 100, 3191–3196 (2003).

    Article  Google Scholar 

  28. 28

    Eklund, M., Axelsson, L., Uhlén, M. & Nygren, P.-Å. Anti-idiotypic protein domains selected from protein A-based affibody libraries. Proteins: Struct. Funct. Genet. 48, 454–462 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Wahlberg, E. et al. An affibody in complex with a target protein: structure and coupled folding. Proc. Natl. Acad. Sci. USA 100, 3185–3190 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Hanes, J., Schaffitzel, C., Knappik, A. & Plückthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Wodak, S.J. & Janin, J. Structural basis of macromolecular recognition. Adv. Protein Chem. 61, 9–73 (2002).

    Article  Google Scholar 

  32. 32

    Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J. & Plückthun, A. Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75–80 (2001).

    CAS  Article  Google Scholar 

  33. 33

    Mian, I.S., Bradwell, A.R. & Olson, A.J. Structure, function and properties of antibody binding sites. J. Mol. Biol. 217, 133–151 (1991).

    CAS  Article  Google Scholar 

  34. 34

    Jeffrey, P.D., Tong, L. & Pavletich, N.P. Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev. 14, 3115–3125 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Sundberg, E.J. & Mariuzza, R.A. Molecular recognition in antibody-antigen complexes. Adv. Protein Chem. 61, 119–160 (2002).

    Article  Google Scholar 

  36. 36

    James, L.C., Roversi, P. & Tawfik, D.S. Antibody multispecificity mediated by conformational diversity. Science 299, 1362–1367 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Xu, L. et al. Directed evolution of high-affinity antibody mimics using mRNA display. Chem. Biol. 9, 933–942 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Zeytun, A., Jeromin, A., Scalettar, B.A., Waldo, G.S. & Bradbury, A.R. Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies. Nat. Biotechnol. 21, 1473–1479 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Cattaneo, A. & Biocca, S. The selection of intracellular antibodies. Trends Biotechnol. 17, 115–121 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Ostermeier, C., Iwata, S., Ludwig, B. & Michel, H. Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. Nat. Struct. Biol. 2, 842–846 (1995).

    CAS  Article  Google Scholar 

  41. 41

    Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: A laboratory manual, edn. 2 (Cold Spring Harbor Press, New York, 1989).

    Google Scholar 

  42. 42

    Myszka, D.G. & Morton, T.A. CLAMP: a biosensor kinetic data analysis program. Trends Biochem. Sci. 23, 149–150 (1998).

    CAS  Article  Google Scholar 

  43. 43

    Collaborative Computational Project, Number 4. The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  44. 44

    Navaza, J. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  45. 45

    Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  Google Scholar 

  46. 46

    Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  47. 47

    McDonald, I.K. & Thornton, J.M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994).

    CAS  Article  Google Scholar 

  48. 48

    Wallace, A.C., Laskowski, R.A. & Thornton, J.M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995).

    CAS  Article  Google Scholar 

  49. 49

    Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    CAS  Article  Google Scholar 

  50. 50

    Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins: Struct. Funct. Genet. 11, 281–296 (1991).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the members of the Plückthun and Grütter laboratories for valuable discussions and David L. Zechel for the critical reading of the manuscript. H.K.B. was supported by a pre-doctoral fellowship of the Roche Research Foundation. M.T.S. was in receipt of a pre-doctoral scholarship from the Fonds der Chemischen Industrie and the Bundesministerium für Bildung und Forschung. This work was supported by the Swiss National Center of Competence in Research in structural biology and the Swiss Krebsliga.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Markus G Grütter or Andreas Plückthun.

Ethics declarations

Competing interests

The technology in this paper has been patented by M.T.S., P.F, H.K.B. and A.P.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Binz, H., Amstutz, P., Kohl, A. et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22, 575–582 (2004). https://doi.org/10.1038/nbt962

Download citation

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