Computational design of environmental sensors for the potent opioid fentanyl

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

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Computational design of an endo-1,4- -xylanase ligand binding site

Protein Engineering Design and Selection ◽

10.1093/protein/gzr006 ◽

2011 ◽

Vol 24 (6) ◽

pp. 503-516 ◽

Cited By ~ 15

Author(s):

A. Morin ◽

K. W. Kaufmann ◽

C. Fortenberry ◽

J. M. Harp ◽

L. S. Mizoue ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Computational Design ◽

Ligand Binding Site

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Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012256 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3708-3718 ◽

Cited By ~ 1

Author(s):

David A. Korasick ◽

Pramod K. Kandoth ◽

John J. Tanner ◽

Melissa G. Mitchum ◽

Lesa J. Beamer

Keyword(s):

Enzyme Activity ◽

Ligand Binding ◽

Binding Site ◽

Crystal Structures ◽

Soybean Cyst Nematode ◽

Kinetic Studies ◽

Cyst Nematode ◽

Serine Hydroxymethyltransferase ◽

Susceptible Cultivar ◽

Soybean Genotype

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5′-phosphate–dependent enzyme that converts l-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Here, we determined five crystal structures of the 1884-residue SHMT8 tetramers from the SCN-susceptible cultivar (cv.) Essex and the SCN-resistant cv. Forrest (whose resistance is derived from the SHMT8 polymorphisms in Peking); the crystal structures were determined in complex with various ligands at 1.4–2.35 Å resolutions. We find that the two Forrest-specific polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the (6S)-tetrahydrofolate–binding site. Ligand-binding and kinetic studies indicate severely reduced affinity for folate and dramatically impaired enzyme activity in Forrest SHMT8. These findings imply widespread effects on folate metabolism in soybean cv. Forrest that have implications for combating the widespread increase in virulent SCN.

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Resetting the ligand binding site of placental protein 13/galectin-13 recovers its ability to bind lactose

Bioscience Reports ◽

10.1042/bsr20181787 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 4

Author(s):

Jiyong Su ◽

Linlin Cui ◽

Yunlong Si ◽

Chenyang Song ◽

Yuying Li ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Crystal Structures ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ligand Binding Site ◽

Wild Type ◽

Placental Protein ◽

High Concentrations ◽

Lower Expression

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to β-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.

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