title: Computational design of ligand-binding proteins with high affinity and selectivity.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Tinberg
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Sagar D
authors.initials: SD
authors.lastname: Khare
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.|Graduate Program in Biological Physics, Structure, and Design, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jiayi
authors.initials: J
authors.lastname: Dou
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Doyle
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jorgen W
authors.initials: JW
authors.lastname: Nelson
authors.affiliation: Institute of Chemical Sciences and Engineering, Institute of Bioengineering, National Centre of Competence in Research (NCCR) of Chemical Biology, École Polytechnique Fédérale de Laussane (EPFL), Laussane, Switzerland.
authors.forename: Alberto
authors.initials: A
authors.lastname: Schena
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Jankowski
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Charalampos G
authors.initials: CG
authors.lastname: Kalodimos
authors.affiliation: Institute of Chemical Sciences and Engineering, Institute of Bioengineering, National Centre of Competence in Research (NCCR) of Chemical Biology, École Polytechnique Fédérale de Laussane (EPFL), Laussane, Switzerland.
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.|Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D001665:Binding Sites; D001709:Biotechnology; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004076:Digoxigenin / Q000737:chemistry / Q000378:metabolism*; D015195:Drug Design; D004958:Estradiol / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D011506:Proteins; D011374:Progesterone; D004958:Estradiol; D004076:Digoxigenin