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Following evolutionary paths to protein-protein interactions with high affinity and selectivity

Nature Structural & Molecular Biology volume 16, pages 1049–1055 (2009)Cite this article

Abstract

How do intricate multi-residue features such as protein-protein interfaces evolve? To address this question, we evolved a new colicin-immunity binding interaction. We started with Im9, which inhibits its cognate DNase ColE9 at 10−14 M affinity, and evolved it toward ColE7, which it inhibits promiscuously (Kd > 10−8 M). Iterative rounds of random mutagenesis and selection toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to an ∼105-fold increase in affinity and a 108-fold increase in selectivity. Analysis of intermediates along the evolved variants revealed that changes in the binding configuration of the Im protein uncovered a latent set of interactions, thus providing the key to the rapid divergence of new Im7 variants. Overall, protein-protein interfaces seem to share the evolvability features of enzymes, that is, the exploitation of promiscuous interactions and alternative binding configurations via 'generalist' intermediates, and the key role of compensatory stabilizing mutations in facilitating the divergence of new functions.

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Figure 1: The presumed genealogy of the in vitro evolution experiment.
Figure 2: Binding configurations of Im7 and Im9 variants.
Figure 3: The latent multipotency of Im9.
Figure 4: Intramolecular stabilizing interactions by residue 37 in wild-type Im9 (a, Val37) and the evolved variant R12-2 (b, Ile37).

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Acknowledgements

We gratefully acknowledge financial support by the Israel Ministry of Science and Technology, the EU training network ProSA and the Sasson and Marjorie Peress Philanthropic Fund. We are grateful to G. Schreiber, O. Cohavi and M. Harel for their help in affinity measurements. C.K. acknowledges the UK Biotechnology and Biological Sciences Research Council for funding.

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Author notes

  1. Anthony H Keeble

    Present address: Present address: Departments of Biochemistry and Infection, Immunity and Inflammation, University of Leicester, Leicester, UK.,

Authors and Affiliations

  1. Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

    Kalia Bernath Levin & Dan S Tawfik

  2. Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel.,

    Kalia Bernath Levin & Shlomo Magdassi

  3. Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, Israel

    Orly Dym & Shira Albeck

  4. Department of Biology (Area 10), University of York, York, UK

    Anthony H Keeble & Colin Kleanthous

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Contributions

K.B.L. performed the selections and variant characterization; O.D. and S.A. crystallized and solved the structures; S.M. participated in the development of the emulsion selections; A.H.K. and C.K. performed stopped-flow measurements; D.S.T. designed the experiments and analyzed the data; K.B.L., O.D. and D.S.T. wrote the manuscript.

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Correspondence to Orly Dym or Dan S Tawfik.

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Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 2983 kb)

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Levin, K., Dym, O., Albeck, S. et al. Following evolutionary paths to protein-protein interactions with high affinity and selectivity. Nat Struct Mol Biol 16, 1049–1055 (2009). https://doi.org/10.1038/nsmb.1670

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