Skip to main content

Thank you for visiting 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.

  • Letter
  • Published:

Natural-like function in artificial WW domains


Protein sequences evolve through random mutagenesis with selection for optimal fitness1. Cooperative folding into a stable tertiary structure is one aspect of fitness, but evolutionary selection ultimately operates on function, not on structure. In the accompanying paper2, we proposed a model for the evolutionary constraint on a small protein interaction module (the WW domain) through application of the SCA, a statistical analysis of multiple sequence alignments3,4. Construction of artificial protein sequences directed only by the SCA showed that the information extracted by this analysis is sufficient to engineer the WW fold at atomic resolution. Here, we demonstrate that these artificial WW sequences function like their natural counterparts, showing class-specific recognition of proline-containing target peptides5,6,7,8. Consistent with SCA predictions, a distributed network of residues mediates functional specificity in WW domains. The ability to recapitulate natural-like function in designed sequences shows that a relatively small quantity of sequence information is sufficient to specify the global energetics of amino acid interactions.

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

Access options

Buy this article

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

Figure 1: Statistical coupling analysis for the WW domain, a proline-binding protein interaction module.
Figure 2: Assays for binding affinity and specificity in WW domains.
Figure 3: A summary of functional measurements for all tested natural and artificial WW domains.
Figure 4: A spatially distributed network underlying WW function.

Similar content being viewed by others


  1. Voigt, C. A., Kauffman, S. & Wang, Z. G. Rational evolutionary design: the theory of in vitro protein evolution. Adv. Protein Chem. 55, 79–160 (2000)

    Article  CAS  Google Scholar 

  2. Socolich, M. et al. Evolutionary information for specifying a protein fold. Nature doi:10.1038/nature03991 (this issue)

  3. Lockless, S. W. & Ranganathan, R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299 (1999)

    Article  CAS  Google Scholar 

  4. Suel, G. M., Lockless, S. W., Wall, M. A. & Ranganathan, R. Evolutionarily conserved networks of residues mediate allosteric communication in proteins. Nature Struct. Biol. 10, 59–69 (2003)

    Article  Google Scholar 

  5. Bedford, M. T., Sarbassova, D., Xu, J., Leder, P. & Yaffe, M. B. A novel pro-Arg motif recognized by WW domains. J. Biol. Chem. 275, 10359–10369 (2000)

    Article  CAS  Google Scholar 

  6. Chen, H. I. & Sudol, M. The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc. Natl Acad. Sci. USA 92, 7819–7823 (1995)

    Article  ADS  CAS  Google Scholar 

  7. Ermekova, K. S. et al. The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled. J. Biol. Chem. 272, 32869–32877 (1997)

    Article  CAS  Google Scholar 

  8. Lu, P. J., Zhou, X. Z., Shen, M. & Lu, K. P. Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283, 1325–1328 (1999)

    Article  ADS  CAS  Google Scholar 

  9. Zarrinpar, A. & Lim, W. A. Converging on proline: the mechanism of WW domain peptide recognition. Nature Struct. Biol. 7, 611–613 (2000)

    Article  CAS  Google Scholar 

  10. Kanelis, V., Rotin, D. & Forman-Kay, J. D. Solution structure of a Nedd4 WW domain-ENaC peptide complex. Nature Struct. Biol. 8, 407–412 (2001)

    Article  CAS  Google Scholar 

  11. Verdecia, M. A., Bowman, M. E., Lu, K. P., Hunter, T. & Noel, J. P. Structural basis for phosphoserine-proline recognition by group IV WW domains. Nature Struct. Biol. 7, 639–643 (2000)

    Article  CAS  Google Scholar 

  12. Kato, Y. et al. Common mechanism of ligand recognition by group II/III WW domains: redefining their functional classification. J. Biol. Chem. 279, 31833–31841 (2004)

    Article  CAS  Google Scholar 

  13. Hu, H. et al. A map of WW domain family interactions. Proteomics 4, 643–655 (2004)

    Article  CAS  Google Scholar 

  14. Otte, L. et al. WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains. Protein Sci. 12, 491–500 (2003)

    Article  CAS  Google Scholar 

  15. Chen, H. I. et al. Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands. J. Biol. Chem. 272, 17070–17077 (1997)

    Article  CAS  Google Scholar 

  16. Espanel, X. & Sudol, M. A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains. J. Biol. Chem. 274, 17284–17289 (1999)

    Article  CAS  Google Scholar 

  17. Kasanov, J., Pirozzi, G., Uveges, A. J. & Kay, B. K. Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities. Chem. Biol. 8, 231–241 (2001)

    Article  CAS  Google Scholar 

  18. Toepert, F., Pires, J. R., Landgraf, C., Oschkinat, H. & Schneider-Mergener, J. Synthesis of an array comprising 837 variants of the hYAP WW protein domain. Angew. Chem. Int. Edn Engl. 40, 897–900 (2001)

    Article  CAS  Google Scholar 

  19. Huang, X. et al. Structure of a WW domain containing fragment of dystrophin in complex with β-dystroglycan. Nature Struct. Biol. 7, 634–638 (2000)

    Article  CAS  Google Scholar 

  20. Carter, P. J., Winter, G., Wilkinson, A. J. & Fersht, A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell 38, 835–840 (1984)

    Article  CAS  Google Scholar 

  21. Hidalgo, P. & MacKinnon, R. Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor. Science 268, 307–310 (1995)

    Article  ADS  CAS  Google Scholar 

  22. Dahiyat, B. I. & Mayo, S. L. De novo protein design: fully automated sequence selection. Science 278, 82–87 (1997)

    Article  CAS  Google Scholar 

  23. Dwyer, M. A., Looger, L. L. & Hellinga, H. W. Computational design of a biologically active enzyme. Science 304, 1967–1971 (2004)

    Article  ADS  CAS  Google Scholar 

  24. Kortemme, T. et al. Computational redesign of protein-protein interaction specificity. Nature Struct. Mol. Biol. 11, 371–379 (2004)

    Article  CAS  Google Scholar 

  25. Kraemer-Pecore, C. M., Lecomte, J. T. & Desjarlais, J. R. A de novo redesign of the WW domain. Protein Sci. 12, 2194–2205 (2003)

    Article  CAS  Google Scholar 

  26. Harbury, P. B., Plecs, J. J., Tidor, B., Alber, T. & Kim, P. S. High-resolution protein design with backbone freedom. Science 282, 1462–1467 (1998)

    Article  CAS  Google Scholar 

  27. Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003)

    Article  ADS  CAS  Google Scholar 

  28. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994)

    Article  CAS  Google Scholar 

  29. Ferguson, N., Johnson, C. M., Macias, M., Oschkinat, H. & Fersht, A. Ultrafast folding of WW domains without structured aromatic clusters in the denatured state. Proc. Natl Acad. Sci. USA 98, 13002–13007 (2001)

    Article  ADS  CAS  Google Scholar 

  30. Delano, W. L. The PyMOL Molecular Graphics System (2002).

Download references


We thank members of the Ranganathan laboratory for advice and critical review of the manuscript, J. P. Noel for providing the pHIS8 expression vector, and K. Voegler for contributing to this project. This study was supported by the Robert A. Welch foundation (R.R.), the Mallinckrodt Foundation Scholar Award (R.R.), NIH grants (M.B.Y.), and a Burroughs-Wellcome Career Development Award (M.B.Y.). D.M.L. was supported by a Howard Hughes Medical Institute pre-doctoral award. W.P.R. is an associate and R.R. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Rama Ranganathan.

Ethics declarations

Competing interests

Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Figure Legends

Text to accompany the below Supplementary Figures. (DOC 35 kb)

Supplementary Figure S1

Binding specificity assays determined by the oriented peptide library assay for (a) 28 natural WW domains and (b) 10 artificial WW domains designed using the SCA matrix. (PDF 1571 kb)

Supplementary Figure S2

Saturation mutagenesis of the peptide ligands for the two major functional classes of WW domains identified. (PDF 6239 kb)

Supplementary Figure S3

Thermodynamic double mutant cycles in the WW domain Nedd4.3 (N39), measuring the energetic coupling between the T28A mutant and mutants at three other sites within the network of co-evolving residues (L3A, E8A, and H23A). (PDF 272 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Russ, W., Lowery, D., Mishra, P. et al. Natural-like function in artificial WW domains. Nature 437, 579–583 (2005).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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