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.
ID: qFpAZe694
Submitter: Paul Chang
Submission Date: June 27, 2018, 11:49 a.m.
Version: 1
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Structure ID | Release Date | Resolution | Structure Title |
---|---|---|---|
1AQB | 1997-07-28T00:00:00+0000 | 1.65 | RETINOL-BINDING PROTEIN (RBP) FROM PIG PLASMA |
1PVX | 1998-10-20T00:00:00+0000 | 1.59 | DO-1,4-BETA-XYLANASE, ROOM TEMPERATURE, PH 4.5 |
2OX1 | 2007-02-19T00:00:00+0000 | 2.33 | Archaeal Dehydroquinase |
1GY7 | 2002-04-21T00:00:00+0000 | 1.6 | N77Y point mutant of S.Cerevisiae NTF2 |
1GYB | 2002-04-22T00:00:00+0000 | 1.9 | N77Y point mutant of yNTF2 bound to FxFG nucleoporin repeat |
1I60 | 2001-03-01T00:00:00+0000 | 1.6 | Structural genomics, IOLI protein |
1I6N | 2001-03-02T00:00:00+0000 | 1.8 | 1.8 A Crystal structure of IOLI protein with a binding zinc atom |
1Z1S | 2005-03-06T00:00:00+0000 | 1.49 | Crystal Structure of Putative Isomerase PA3332 from Pseudomonas aeruginosa |
4J8T | 2013-02-14T00:00:00+0000 | 2.05 | Engineered Digoxigenin binder DIG10.2 |
4J9A | 2013-02-15T00:00:00+0000 | 3.2 | Engineered Digoxigenin binder DIG10.3 |
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 |