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.

Submission Details

ID: qFpAZe694

Submitter: Paul Chang

Submission Date: June 27, 2018, 11:49 a.m.

Version: 1

Publication Details
Tinberg CE;Khare SD;Dou J;Doyle L;Nelson JW;Schena A;Jankowski W;Kalodimos CG;Johnsson K;Stoddard BL;Baker D,Nature (2013) Computational design of ligand-binding proteins with high affinity and selectivity. PMID:24005320
Additional Information

Number of data points 1728
Proteins Retinol-binding protein 4 ; putative gluconolactonase in protein family PF08450 ; 3-dehydroquinate dehydratase ; cystatin-like fold protein (bxe_b1374) ; nuclear transport factor 2 (Ntf2) ; Inosose isomerase ; PUTATIVE SNOAL-LIKE POLYKETIDE CYCLASE [CARBOHYDRATE PHOSPHATASE] (MLR7391) ; Putative calcium/calmodulin-dependent protein kinase type II association domain (YP_315894.1) ; domain of unknown function with a cystatin-like fold (npun_r1993) ; Uncharacterized PhzA/B-like protein PA3332 ; phenazine biosynthesis ; Endo-1,4-beta-xylanase ; Beta-glucanase ; Nuclear transport factor 2
Unique complexes 767
Assays/Quantities/Protocols Experimental Assay: Input DIG10.1L library deep sequencing counts ; Experimental Assay: Selected DIG10.1L library deep sequencing counts ; Experimental Assay: Kd of DIG10.3 variants for DIG-PEG3-Alexa488 measured by fluorescence polarization ; Experimental Assay: β-estradiol IC50 measured by fluorescence polarization equilibrium competition ; Experimental Assay: progesterone IC50 measured by fluorescence polarization equilibrium competition ; Experimental Assay: digitoxigenin IC50 measured by fluorescence polarization equilibrium competition ; Experimental Assay: DIG IC50 measured by fluorescence polarization equilibrium competition ; Experimental Assay: digoxin IC50 measured by fluorescence polarization equilibrium competition ; Experimental Assay: -TΔS determined by isothermal titration calorimetry ; Experimental Assay: ΔH determined by isothermal titration calorimetry ; Experimental Assay: ΔG determined by isothermal titration calorimetry ; Experimental Assay: Kd determined by isothermal titration calorimetry ; Experimental Assay: Binding Signal with DIG-BSA-biotin ; Experimental Assay: Binding Signal with DIG-RNase-biotin ; Experimental Assay: Yeast surface protein expression ; Derived Quantity: ΔΔG (DIG10.3 – Variant) ; Derived Quantity: ΔΔG (DIG – digoxin) ; Derived Quantity: β-estradiol Ki ; Derived Quantity: ΔΔG (DIG – β-estradiol) ; Derived Quantity: digitoxigenin Ki ; Derived Quantity: ΔΔG (DIG – progesterone) ; Derived Quantity: digoxin Ki ; Derived Quantity: ΔΔG (DIG – digitoxigenin) ; Derived Quantity: DIG Ki ; Derived Quantity: progesterone Ki
Libraries Features of ordered designs ; Thermodynamic parameters of DIG binding to DIG10 variants determined by isothermal titration calorimetry ; DIG10.1L library deep sequencing counts ; DIG10.3 steroid binding affinities measured by fluorescence polarization equilibrium competition experiments ; Binding affinities of DIG10.3 variants for DIG-PEG3-Alexa488 measured by fluorescence polarization
Sequence Assay Result Units