scholarly journals New Insights of the Zn(II)-Induced P2 × 4R Positive Allosteric Modulation: Role of Head Receptor Domain SS2/SS3, E160 and D170

2020 ◽  
Vol 21 (18) ◽  
pp. 6940
Author(s):  
Francisco Andrés Peralta ◽  
J. Pablo Huidobro-Toro
Keyword(s):  
Amino Acid ◽  
Disulfide Bonds ◽  
Allosteric Modulation ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Electrophysiological Recordings ◽  
Poisson Boltzmann ◽  
Type Receptor ◽  
Receptor Domain

P2 × 4R is allosterically modulated by Zn(II), and despite the efforts to understand the mechanism, there is not a consensus proposal; C132 is a critical amino acid for the Zn(II) modulation, and this residue is located in the receptor head domain, forming disulfide SS3. To ascertain the role of the SS2/SS3 microenvironment on the rP2 × 4R Zn(II)-induced allosteric modulation, we investigated the contribution of each individual SS2/SS3 cysteine plus carboxylic acid residues E118, E160, and D170, located in the immediate vicinity of the SS2/SS3 disulfide bonds. To this aim, we combined electrophysiological recordings with protein chemical alkylation using thiol reagents such as N-ethylmaleimide or iodoacetamide, and a mutation of key amino acid residues together with P2 × 4 receptor bioinformatics. P2 × 4R alkylation in the presence of the metal obliterated the allosteric modulation, a finding supported by the site-directed mutagenesis of C132 and C149 by a corresponding alanine. In addition, while E118Q was sensitive to Zn(II) modulation, the wild type receptor, mutants E160Q and D170N, were not, suggesting that these acid residues participate in the modulatory mechanism. Poisson–Boltzmann analysis indicated that the E160Q and D170N mutants showed a shift towards more positive electrostatic potential in the SS2/SS3 microenvironment. Present results highlight the role of C132 and C149 as putative Zn(II) ligands; in addition, we infer that acid residues E160 and D170 play a role attracting Zn(II) to the head receptor domain.

scholarly journals Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis

1997 ◽  
Vol 323 (1) ◽  
pp. 61-64 ◽  
Author(s):  
Kazuya MATSUURA ◽  
Yoshihiro DEYASHIKI ◽  
Kumiko SATO ◽  
Naoko ISHIDA ◽  
Gunpei MIWA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Dihydrodiol Dehydrogenase ◽  
Inactive Protein

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20α-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3α-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

Functional roles of cationic amino acid residues in the sodium-dicarboxylate cotransporter 3 (NaDC-3) from winter flounder

2006 ◽  
Vol 291 (6) ◽  
pp. F1224-F1231 ◽  
Author(s):  
Yohannes Hagos ◽  
Jürgen Steffgen ◽  
Ahsan N. Rizwan ◽  
Denis Langheit ◽  
Ariane Knoll ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Helix ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cationic Amino Acid ◽  
Functional Roles ◽  
Inward Currents ◽  
Induced Currents ◽  
Positively Charged

In the present study, we determined the functional role of 15 positively charged amino acid residues at or within 1 of the predicted 11 transmembrane helixes of the flounder renal sodium-dicarboxylate cotransporter fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K), and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [14C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, Km and Δ Imax values comparable to wild-type (WT) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed but showed small transport rates and succinate-induced currents. K114I, located within transmembrane helix 4, showed [14C]succinate uptake similar to WT but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [14C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than WT, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K114I, K114Q) than, those evoked by WT. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium-dicarboxylate cotransport.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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