Computational design of ligand-binding proteins with high affinity and selectivity

Nature. 2013 Sep 12;501(7466):212-216. doi: 10.1038/nature12443. Epub 2013 Sep 4.


The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Biotechnology
  • Computer Simulation*
  • Crystallography, X-Ray
  • Digoxigenin / chemistry
  • Digoxigenin / metabolism*
  • Drug Design*
  • Estradiol / chemistry
  • Estradiol / metabolism
  • Ligands
  • Models, Molecular
  • Progesterone / chemistry
  • Progesterone / metabolism
  • Protein Binding
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Reproducibility of Results
  • Substrate Specificity


  • Ligands
  • Proteins
  • Progesterone
  • Estradiol
  • Digoxigenin

Associated data

  • PDB/4J8T
  • PDB/4J9A