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

Study Summary

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

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
396.8 A,B,C,D Nuclear transport factor 2 P33331 NTF2_YEAST
368.0 A,B,C,D Nuclear transport factor 2 Q6FRC6 NTF2_CANGA
361.6 A,B,C,D Nuclear transport factor 2 Q75AA5 NTF2_ASHGO
100.0 Beta-glucanase P17989 GUB_FIBSS
100.0 Endo-1,4-beta-xylanase P81536 XYNA_BYSSP
100.0 Uncharacterized PhzA/B-like protein PA3332 Q9HYR3 Y3332_PSEAE
100.0 Inosose isomerase P42419 IOLI_BACSU
400.0 A,B,C,D 3-dehydroquinate dehydratase O30011 AROD_ARCFU
197.8 A,B Retinol-binding protein 4 P02753 RET4_HUMAN
197.8 A,B Retinol-binding protein 4 P61641 RET4_PANTR
189.0 A,B Retinol-binding protein 4 M5AXY1 RET4_FELCA
189.0 A,B Retinol-binding protein 4 Q28369 RET4_HORSE
185.8 A,B Retinol-binding protein 4 P27485 RET4_PIG
181.8 A,B Retinol-binding protein 4 P06912 RET4_RABIT
184.6 A,B Retinol-binding protein 4 P18902 RET4_BOVIN