scholarly journals Ethanol Inhibition ofN-Methyl-d-aspartate Receptors Is Reduced by Site-directed Mutagenesis of a Transmembrane Domain Phenylalanine Residue

2001 ◽  
Vol 276 (48) ◽  
pp. 44729-44735 ◽  
Author(s):  
Kimberly M. Ronald ◽  
Tooraj Mirshahi ◽  
John J. Woodward
Keyword(s):  
Transmembrane Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ethanol Inhibition ◽  
Phenylalanine Residue

scholarly journals Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

2003 ◽  
Vol 371 (2) ◽  
pp. 443-449 ◽  
Author(s):  
Frank NEUSCHÄFER-RUBE ◽  
Eva ENGEMAIER ◽  
Sina KOCH ◽  
Ulrike BÖER ◽  
Gerhard P. PÜSCHEL
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Plasma Membrane Receptors

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

scholarly journals Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

2019 ◽  
Vol 44 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Jean-Baptiste Chéron ◽  
Amanda Soohoo ◽  
Yi Wang ◽  
Jérôme Golebiowski ◽  
Serge Antonczak ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Taste Receptor ◽  
Receptor Activation ◽  
Sweet Taste ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

scholarly journals Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

1999 ◽  
Vol 344 (1) ◽  
pp. 205-209 ◽  
Author(s):  
Ana M. PAJOR ◽  
Sally J. KRAJEWSKI ◽  
Nina SUN ◽  
Rama GANGULA
Keyword(s):  
Protein Trafficking ◽  
Direct Relationship ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

scholarly journals LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor

2016 ◽  
Vol 82 (17) ◽  
pp. 5364-5374 ◽  
Author(s):  
Marija Miljkovic ◽  
Gordana Uzelac ◽  
Nemanja Mirkovic ◽  
Giulia Devescovi ◽  
Dzung B. Diep ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Sugar Transport ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacteriocin Activity ◽  
Binding Studies ◽  
Content Type ◽  
The Third

ABSTRACTThe Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. AlthoughyvjBis highly conserved in the genusLactococcus, the bacteriocin appears to be active only against the subspeciesL. lactissubsp.lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353).In vitroexperiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG.In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.IMPORTANCEThe antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr356and Ala353residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

scholarly journals A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

2010 ◽  
Vol 299 (5) ◽  
pp. F1111-F1119 ◽  
Author(s):  
María Castañeda-Bueno ◽  
Norma Vázquez ◽  
Ismael Bustos-Jaimes ◽  
Damian Hernández ◽  
Erika Rodríguez-Lobato ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Transmembrane Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Transmembrane Region ◽  
Protein Target ◽  
The Difference ◽  
Alignment Analysis

Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na+-Cl− cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8–12 contains the residues that produce this difference. Here, an alignment analysis of TM 8–12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using 22Na+ uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties.

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

1996 ◽  
Vol 1 (6) ◽  
pp. 542-550 ◽  
Author(s):  
Lígia M. Saraiva ◽  
Carlos A. Salgueiro ◽  
Jean LeGall ◽  
Walter M. A. M. van Dongen ◽  
António V. Xavier
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cytochromes C ◽  
Phenylalanine Residue

scholarly journals Cysteine 144 in the Third Transmembrane Domain of the Creatine Transporter Is Located Close to a Substrate-binding Site

2001 ◽  
Vol 276 (50) ◽  
pp. 46983-46988 ◽  
Author(s):  
Joanna R. Dodd ◽  
David L. Christie
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Creatine Transporter ◽  
The Third ◽  
Low Concentrations ◽  
Rapid Inactivation

All creatine transporters contain a cysteine residue (Cys144) in the third transmembrane domain that is not present in other members of the Na+,Cl−-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys144for transporter function. Replacement of Cys144with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higherKm(0.33 compared with 0.18 mm). Substitution of Cys144with Leu gave a mutant with a 5-fold higherKmand a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys144was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+and Cl−ions were not necessary for MTSEA inactivation, but Na+was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.

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