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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Halabi, N., Rivoire, O., Leibler, S. & Ranganathan, R. Protein sectors: evolutionary units of three-dimensional structure. Cell 138, 774–786 (2009)
Lockless, S. W. & Ranganathan, R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299 (1999)
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)
Socolich, M. et al. Evolutionary information for specifying a protein fold. Nature 437, 512–518 (2005)
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)
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)
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)
Draghi, J. A., Parsons, T. L., Wagner, G. P. & Plotkin, J. B. Mutational robustness can facilitate adaptation. Nature 463, 353–355 (2010)
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)
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)
Anfinsen, C. B. Principles that govern the folding of protein chains. Science 181, 223–230 (1973)
Reynolds, K. A., McLaughlin, R. N. & Ranganathan, R. Hot spots for allosteric regulation on protein surfaces. Cell 147, 1564–1575 (2011)
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)
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)
Shulman, A. I., Larson, C., Mangelsdorf, D. J. & Ranganathan, R. Structural determinants of allosteric ligand activation in RXR heterodimers. Cell 116, 417–429 (2004)
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)
Bradley, P., Misura, K. M. S. & Baker, D. Toward high-resolution de novo structure prediction for small proteins. Science 309, 1868–1871 (2005)
Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003)
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)
Chi, C. N. et al. Reassessing a sparse energetic network within a single protein domain. Proc. Natl Acad. Sci. USA 105, 4679–4684 (2008)
Dove, S. L., Joung J. K & Hochschild, A. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386, 627–630 (1997)
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)
Adkar, B. V. et al. Protein model discrimination using mutational sensitivity derived from deep sequencing. Structure 20, 371–381 (2012)
Fowler, D. M. et al. High-resolution mapping of protein sequence-function relationships. Nature Methods 7, 741–746 (2010)
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)
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)
Whipple, F. W., Ptashne, M. & Hochschild, A. The activation defect of a λ-cI positive control mutant. J. Mol. Biol. 265, 261–265 (1997)
Clackson, T. & Wells J. A A hot spot of binding energy in a hormone-receptor interface. Science 267, 383–386 (1995)
Songyang, Z. et al. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275, 73–77 (1997)
Kussell, E. & Leibler, S. Phenotypic diversity, population growth, and information in fluctuating environments. Science 309, 2075–2078 (2005)
Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)
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)
Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000)
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)
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)
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.).
The authors declare no competing financial interests.
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) doi:10.1038/nature11500
Physical Review E (2019)
Mutational analysis of a catalytically important loop containing active site and substrate-binding site in Escherichia coli phytase AppA
Bioscience, Biotechnology, and Biochemistry (2019)
Chemical Reviews (2019)
In silico identification and design of potent peptide inhibitors against PDZ-3 domain of Postsynaptic Density Protein (PSD-95)
Journal of Biomolecular Structure and Dynamics (2019)