Letter | Published:

Manipulation of ligand binding affinity by exploitation of conformational coupling

Nature Structural Biology volume 8, pages 795798 (2001) | Download Citation

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Abstract

Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from a 100-fold improvement (7.4 nM) to a two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).

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Acknowledgements

The authors would like to thank E.J. Toone for helpful discussions regarding the thermodynamics of maltose binding. This work was funded by grants from the National Institutes of Health and the Office of Naval Research.

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Author notes

    • Jonathan S. Marvin

    Present address: Department of Protein Engineering, Genentech Inc., South San Fransicso, California 94080, USA.

Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

    • Jonathan S. Marvin
    •  & Homme W. Hellinga

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Correspondence to Homme W. Hellinga.

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DOI

https://doi.org/10.1038/nsb0901-795

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