The ligand binding site of the neurokinin 2 receptor. Site-directed mutagenesis and identification of neurokinin A binding residues in the human neurokinin 2 receptor.

Abstract The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 1010m−1). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded β-barrel domain encompasses the ligand-binding site. We have now characterized the T4 binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the β-barrel motif. Mutations within this region (Val228Glu, Cys234Trp, Thr235Trp, Thr235Gln, Lys253Ala, and Lys253Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T4 binding. However, binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu246Thr) without compromising the proper folding of this mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within theβ -barrel. The importance of the binding site’s aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe249 in the middle of strand 3B completely abolished T4 binding, while the substitution of several other phenylalanines had no effect.

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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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