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.

The spatial architecture of protein function and adaptation


Statistical analysis of protein evolution suggests a design for natural proteins in which sparse networks of coevolving amino acids (termed sectors) comprise the essence of three-dimensional structure and function1,2,3,4,5. However, proteins are also subject to pressures deriving from the dynamics of the evolutionary process itself—the ability to tolerate mutation and to be adaptive to changing selection pressures6,7,8,9,10. To understand the relationship of the sector architecture to these properties, we developed a high-throughput quantitative method for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid. Using a PDZ domain (PSD95pdz3) model system, we show that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution. In addition, we find that adaptation to a new binding specificity initiates exclusively through variation within sector residues. A combination of just two sector mutations located near and away from the ligand-binding site suffices to switch the binding specificity of PSD95pdz3 quantitatively towards a class-switching ligand. The localization of functional constraint and adaptive variation within the sector has important implications for understanding and engineering proteins.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Sector architecture in the PDZ domain family.
Figure 2: Complete single mutagenesis in PSD95pdz3.
Figure 3: The relationship of mutational sensitivity of positions to the protein sector.
Figure 4: Adaptation through sector variation.


  1. 1

    Halabi, N., Rivoire, O., Leibler, S. & Ranganathan, R. Protein sectors: evolutionary units of three-dimensional structure. Cell 138, 774–786 (2009)

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Russ, W. P., Lowery, D. M., Mishra, P., Yaffe, M. B. & Ranganathan, R. Natural-like function in artificial WW domains. Nature 437, 579–583 (2005)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Socolich, M. et al. Evolutionary information for specifying a protein fold. Nature 437, 512–518 (2005)

    ADS  CAS  Article  Google Scholar 

  5. 5

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

    Article  Google Scholar 

  6. 6

    Bershtein, S., Segal, M., Bekerman, R., Tokuriki, N. & Tawfik, D. S. Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein. Nature 444, 929–932 (2006)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Bloom, J. D., Labthavikul, S. T., Otey, C. R. & Arnold, F. H. Protein stability promotes evolvability. Proc. Natl Acad. Sci. USA 103, 5869–5874 (2006)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Draghi, J. A., Parsons, T. L., Wagner, G. P. & Plotkin, J. B. Mutational robustness can facilitate adaptation. Nature 463, 353–355 (2010)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Tiana, G., Shakhnovich, B. E., Dokholyan, N. V. & Shakhnovich, E. I. Imprint of evolution on protein structures. Proc. Natl Acad. Sci. USA 101, 2846–2851 (2004)

    ADS  CAS  Article  Google Scholar 

  10. 10

    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)

    CAS  Article  Google Scholar 

  11. 11

    Anfinsen, C. B. Principles that govern the folding of protein chains. Science 181, 223–230 (1973)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Reynolds, K. A., McLaughlin, R. N. & Ranganathan, R. Hot spots for allosteric regulation on protein surfaces. Cell 147, 1564–1575 (2011)

    CAS  Article  Google Scholar 

  13. 13

    Peterson, F. C., Penkert, R. R., Volkman, B. F. & Prehoda, K. E. Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition. Mol. Cell 13, 665–676 (2004)

    CAS  Article  Google Scholar 

  14. 14

    Hatley, M. E., Lockless, S. W., Gibson, S. K., Gilman, A. G. & Ranganathan, R. Allosteric determinants in guanine nucleotide-binding proteins. Proc. Natl Acad. Sci. USA 100, 14445–14450 (2003)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Shulman, A. I., Larson, C., Mangelsdorf, D. J. & Ranganathan, R. Structural determinants of allosteric ligand activation in RXR heterodimers. Cell 116, 417–429 (2004)

    CAS  Article  Google Scholar 

  16. 16

    Smock, R. R. O., Russ, W. P., Swain, J. F., Leibler, S., Ranganathan, R. & Gierasch, L. M. An interdomain sector mediating allostery in Hsp70 molecular chaperones. Mol. Syst. Biol. 6, 414 (2010)

    Article  Google Scholar 

  17. 17

    Bradley, P., Misura, K. M. S. & Baker, D. Toward high-resolution de novo structure prediction for small proteins. Science 309, 1868–1871 (2005)

    ADS  CAS  Article  Google Scholar 

  18. 18

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

    ADS  CAS  Article  Google Scholar 

  19. 19

    Hilser, V. J. & Thompson, E. B. Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins. Proc. Natl Acad. Sci. USA 104, 8311–8315 (2007)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Chi, C. N. et al. Reassessing a sparse energetic network within a single protein domain. Proc. Natl Acad. Sci. USA 105, 4679–4684 (2008)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Dove, S. L., Joung J. K & Hochschild, A. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386, 627–630 (1997)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Niethammer, M. et al. CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90. Neuron 20, 693–707 (1998)

    CAS  Article  Google Scholar 

  23. 23

    Adkar, B. V. et al. Protein model discrimination using mutational sensitivity derived from deep sequencing. Structure 20, 371–381 (2012)

    CAS  Article  Google Scholar 

  24. 24

    Fowler, D. M. et al. High-resolution mapping of protein sequence-function relationships. Nature Methods 7, 741–746 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Kinney, J. B., Murugan, A., Callan, C. G. & Cox, E. C. Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence. Proc. Natl Acad. Sci. USA 107, 9158–9163 (2010)

    ADS  CAS  Article  Google Scholar 

  26. 26

    van Opijnen, T., Bodi, K. L. & Camilli, A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nature Methods 6, 767–772 (2009)

    CAS  Article  Google Scholar 

  27. 27

    Whipple, F. W., Ptashne, M. & Hochschild, A. The activation defect of a λ-cI positive control mutant. J. Mol. Biol. 265, 261–265 (1997)

    CAS  Article  Google Scholar 

  28. 28

    Clackson, T. & Wells J. A A hot spot of binding energy in a hormone-receptor interface. Science 267, 383–386 (1995)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Songyang, Z. et al. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275, 73–77 (1997)

    CAS  Article  Google Scholar 

  30. 30

    Kussell, E. & Leibler, S. Phenotypic diversity, population growth, and information in fluctuating environments. Science 309, 2075–2078 (2005)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)

    CAS  Article  Google Scholar 

  32. 32

    Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997)

    CAS  Article  Google Scholar 

  33. 33

    Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Bushman, F. D., Shang, C. & Ptashne, M. A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor. Cell 58, 1163–1171 (1989)

    CAS  Article  Google Scholar 

  35. 35

    Jain, D., Nickels, B. E., Sun, L., Hochschild, A. & Darst, S. A. Structure of a ternary transcription activation complex. Mol. Cell 13, 45–53 (2004)

    CAS  Article  Google Scholar 

Download references


We thank W. Russ, P. Mishra and other members of the Ranganathan laboratory for contributions to this work, W. Wakeland and C. Liang for assistance with Solexa sequencing, E. Curry and A. Mobley for assistance with flow cytometry, and M. Elowitz for providing the pZ plasmids. We acknowledge support from the University of Texas Southwestern Graduate School and Pharmacology Training Grant (R. N. M.), the Helen Hay Whitney Fellowship program (F.J.P.) and support from the National Institutes of Health (R01EY018720-05, R.R.), The Robert A. Welch Foundation (I-1366, R.R.) and the Green Center for Systems Biology (R.R.).

Author information




R.N.M. and R.R. developed the research plan and experimental strategy. R.N.M. built the B2H assay, collected the data and wrote and executed the code for analysis of the B2H and sequencing data. F.J.P. and W.S.G. improved the dynamic range of the B2H assay. A.R. carried out the mutational analysis in Fig. 4 e, f. R.N.M. and R.R. analysed the data and wrote the paper.

Corresponding author

Correspondence to Rama Ranganathan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7, Supplementary Data and Supplementary References. (PDF 2404 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

McLaughlin Jr, R., Poelwijk, F., Raman, A. et al. The spatial architecture of protein function and adaptation. Nature 491, 138–142 (2012).

Download citation

Further reading


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