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 PDB Entries

Structure ID Release Date Resolution Structure Title
1KT6 2003-06-03 1.1 Crystal structure of bovine holo-RBP at pH 9.0
1KT7 2003-06-03 1.27 Crystal structure of bovine holo-RBP at pH 7.0
3H0O 2010-04-21 1.4 The importance of CH-Pi stacking interactions between carbohydrate and aromatic residues in truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase
1KT3 2003-06-03 1.4 Crystal structure of bovine holo-RBP at pH 2.0
1KT5 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 4.0
1KT4 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 3.0
1Z1S 2005-04-19 1.49 Crystal Structure of Putative Isomerase PA3332 from Pseudomonas aeruginosa
5NU9 2018-03-07 1.5 Structure of human amniotic fluid RBP4 saturated with palmitate
5NU2 2018-03-07 1.5 Structure of non-fluorescent human urine RBP4
5NU7 2018-03-07 1.5 Structure of human holo plasma RBP4
3AXE 2012-02-15 1.53 The truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase V18Y/W203Y in complex with cellotetraose (cellobiose density was observed)
3AXD 2012-02-15 1.53 The truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase V18Y/W203Y in apo-form
1PVX 1999-10-20 1.59 DO-1,4-BETA-XYLANASE, ROOM TEMPERATURE, PH 4.5
5NU8 2018-03-07 1.59 Structure of human urine RBP4 saturated with palmitate
5NUA 2018-03-07 1.6 Structure of human urine RBP4 saturated with laurate
1I60 2002-03-13 1.6 Structural genomics, IOLI protein
5NUB 2018-03-14 1.6 Structure of human amniotic fluid RBP4 saturated with laurate
1GY7 2002-06-20 1.6 N77Y point mutant of S.Cerevisiae NTF2
2WQ9 2010-09-01 1.65 Crystal Structure of RBP4 bound to Oleic Acid
1AQB 1998-01-28 1.65 RETINOL-BINDING PROTEIN (RBP) FROM PIG PLASMA
5NU6 2018-03-07 1.68 Structure of non-fluorescent human amniotic fluid RBP4
3HR9 2009-07-07 1.7 The truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase F40I mutant
1MVE 2003-07-15 1.7 Crystal structure of a natural circularly-permutated jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes
1HBQ 1994-01-31 1.7 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1JYD 2003-07-01 1.7 Crystal Structure of Recombinant Human Serum Retinol-Binding Protein at 1.7 A Resolution
1I6N 2002-03-13 1.8 1.8 A Crystal structure of IOLI protein with a binding zinc atom
2WR6 2010-09-15 1.8 Structure of the complex of RBP4 with linoleic acid
1FEL 1994-11-01 1.8 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1ERB 1994-01-31 1.9 THE INTERACTION OF N-ETHYL RETINAMIDE WITH PLASMA RETINOL-BINDING PROTEIN (RBP) AND THE CRYSTAL STRUCTURE OF THE RETINOID-RBP COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1HBP 1994-01-31 1.9 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1FEN 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1FEM 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1GYB 2002-06-20 1.9 N77Y point mutant of yNTF2 bound to FxFG nucleoporin repeat
5NTY 2018-03-07 2.0 Structure of non-fluorescent human plasma RBP4
1RBP 1991-07-15 2.0 CRYSTALLOGRAPHIC REFINEMENT OF HUMAN SERUM RETINOL BINDING PROTEIN AT 2 ANGSTROMS RESOLUTION
1JYJ 2003-07-01 2.0 Crystal Structure of a Double Variant (W67L/W91H) of Recombinant Human Serum Retinol-binding Protein at 2.0 A Resolution
4J8T 2013-06-26 2.05 Engineered Digoxigenin binder DIG10.2
2R49 2008-09-02 2.2 Mutational and Structural Studies of E85I Reveal the Flexible Loops of Fibrobacter succinogenes 1,3-1,4-beta-D-GlucanaseGlucanase
4O9S 2014-07-02 2.3 Crystal structure of Retinol-Binding Protein 4 (RBP4)in complex with a non-retinoid ligand
1ZM1 2006-05-10 2.3 Crystal structures of complex F. succinogenes 1,3-1,4-beta-D-glucanase and beta-1,3-1,4-cellotriose
2OX1 2008-02-19 2.33 Archaeal Dehydroquinase
4PSQ 2014-07-02 2.4 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with a non-retinoid ligand
1BRQ 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
1BRP 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
2WQA 2010-09-01 2.85 Complex of TTR and RBP4 and Oleic Acid
3FMZ 2009-01-27 2.9 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with non-retinoid ligand
1RLB 1996-04-11 3.1 RETINOL BINDING PROTEIN COMPLEXED WITH TRANSTHYRETIN
1QAB 1999-02-16 3.2 The structure of human retinol binding protein with its carrier protein transthyretin reveals interaction with the carboxy terminus of RBP
4J9A 2013-06-26 3.2 Engineered Digoxigenin binder DIG10.3
3BSZ 2008-11-11 3.38 Crystal structure of the transthyretin-retinol binding protein-Fab complex

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