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 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 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.

scholarly journals A novel activating mutation in the thyrotropin receptor gene in an autonomously functioning thyroid nodule developed by a Japanese patient

2000 ◽  
pp. 471-477 ◽  
Author(s):  
S Kosugi ◽  
N Hai ◽  
H Okamoto ◽  
H Sugawa ◽  
T Mori
Keyword(s):  
Japanese Patient ◽  
Transmembrane Helix ◽  
Japanese Patients ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Thyrotropin Receptor ◽  
Functional Studies ◽  
Activating Mutations ◽  
The Third ◽  
Activating Mutation

OBJECTIVE: A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. DESIGN: A typical AFTN developed in a 69-year-old Japanese woman was studied. METHODS: The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. RESULTS: We identified a novel heterozygous TSHR gene mutation, Leu512-->Arg (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, confirming that the mutation was the direct cause of the AFTN. TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505-513 revealed that mutations involving residues other than these three did not show constitutive activation. CONCLUSION: This is the first TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with another helix.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Determination of the Catalytic Base in Family 48 Glycosyl Hydrolases

2011 ◽  
Vol 77 (17) ◽  
pp. 6274-6276 ◽  
Author(s):  
Maxim Kostylev ◽  
David B. Wilson
Keyword(s):  
Aspartic Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Thermobifida Fusca ◽  
Glycosyl Hydrolases ◽  
Content Type ◽  
Modeling Data ◽  
Partial Activity

ABSTRACTThe catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base inThermobifida fuscaCel48A is aspartic acid 225. Of the tested mutants, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.

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