scholarly journals Identification of Amino Acid Residues Important for Ligand Binding to Fas

1997 ◽  
Vol 185 (8) ◽  
pp. 1487-1492 ◽  
Author(s):  
Gary C. Starling ◽  
Jürgen Bajorath ◽  
John Emswiler ◽  
Jeffrey A. Ledbetter ◽  
Alejandro Aruffo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Immune Responses ◽  
Ligand Binding ◽  
Fusion Proteins ◽  
Molecular Model ◽  
Tumor Necrosis Factor Receptor ◽  
Jurkat Cells ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Necrosis Factor

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas–FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore™ assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

scholarly journals Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells

2020 ◽  
Vol 13 (614) ◽  
pp. eaax5647 ◽  
Author(s):  
Christos Karathanasis ◽  
Juliane Medler ◽  
Franziska Fricke ◽  
Sonja Smith ◽  
Sebastian Malkusch ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cancer Progression ◽  
Single Molecule ◽  
Tumor Necrosis Factor Receptor ◽  
Wild Type ◽  
Oligomeric State ◽  
Superresolution Microscopy ◽  
Ligand Free ◽  
Light Chain Enhancer ◽  
Necrosis Factor

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

scholarly journals Role of TNFR1 in the innate airway hyperresponsiveness of obese mice

2012 ◽  
Vol 113 (9) ◽  
pp. 1476-1485 ◽  
Author(s):  
Ming Zhu ◽  
Alison S. Williams ◽  
Lucas Chen ◽  
Allison P. Wurmbrand ◽  
Erin S. Williams ◽  
...  
Keyword(s):  
Airway Hyperresponsiveness ◽  
Bronchoalveolar Lavage Fluid ◽  
Pulmonary Inflammation ◽  
Tumor Necrosis Factor Receptor ◽  
Lavage Fluid ◽  
Forced Oscillation Technique ◽  
Obese Mice ◽  
Wild Type ◽  
Necrosis Factor

The purpose of this study was to examine the role of tumor necrosis factor receptor 1 (TNFR1) in the airway hyperresponsiveness characteristic of obese mice. Airway responsiveness to intravenous methacholine was measured using the forced oscillation technique in obese Cpe fat mice that were either sufficient or genetically deficient in TNFR1 ( Cpe fat and Cpe fat/TNFR1−/− mice) and in lean mice that were either sufficient or genetically deficient in TNFR1 [wild-type (WT) and TNFR1−/− mice]. Compared with lean WT mice, Cpe fat mice exhibited airway hyperresponsiveness. Airway hyperresponsives was also greater in Cpe fat/TNFR1−/− than in Cpe fat mice. Compared with WT mice, Cpe fat mice had increases in bronchoalveolar lavage fluid concentrations of several inflammatory moieties including eotaxin, IL-9, IP-10, KC, MIG, and VEGF. These factors were also significantly elevated in Cpe fat/TNFR1−/− vs. TNFR1−/− mice. Additional moieties including IL-13 were also elevated in Cpe fat/TNFR1−/− vs. TNFR1−/− mice but not in Cpe fat vs. WT mice. IL-17A mRNA expression was greater in Cpe fat/TNFR1−/− vs. Cpe fat mice and in TNFR1−/− vs. WT mice. Analysis of serum indicated that obesity resulted in systemic as well as pulmonary inflammation, but TNFR1 deficiency had little effect on this systemic inflammation. Our results indicate that TNFR1 is protective against the airway hyperresponsiveness associated with obesity and suggest that effects on pulmonary inflammation may be contributing to this protection.

Model of a folded polypeptide chain

1950 ◽  
Vol 137 (886) ◽  
pp. 79-79
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Molecular Model ◽  
Polymer Chains ◽  
Side Chains ◽  
Amino Acid Residues ◽  
Infra Red ◽  
New Type ◽  
Folded Proteins ◽  
Red Radiation

The models on view in the ante-room show a way of folding a polypeptide chain which is consistent with some observations we have recently made with polarized infra-red radiation (Ambrose & Hanby 1949; Ambrose, Elliott & Temple 1949). The α -folded proteins, keratin, myosin and tropomyosin, have been found when oriented to show greater absorption of the N-H frequency when the electric vector of the absorbed radiation is in the direction of the fibre axis, hence the N-H bond must be preferentially oriented in this direction. A study of models has suggested that the only likely folding of the polypeptide chain consistent with this fact involves a seven-membered ring containing two amino-acid residues; the ring is completed by hydrogen bonds: A new type of atomic model which has been developed in our laboratories has been used. The scale is 0·8 in. to the Angstrom unit. The valency links, while allowing free rotation about single co-valent bonds, also allow some distortion of the bond angles when strains occur but are strong enough to allow long polymer chains to be built. The molecular model exhibited shows twenty-four amino-acid residues, with side chains on one side of the back-bone, representative of those occurring in myosin; the side chains on the other side have been removed for clearness and their positions indicated by single carbon atoms.

scholarly journals The role of amino acid α38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland

1999 ◽  
Vol 343 (3) ◽  
pp. 681-685 ◽  
Author(s):  
Tao ZHENG ◽  
Thomas BRITTAIN ◽  
Nicholas J. WATMOUGH ◽  
Roy E. WEBER
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Significant Contribution ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Oxygen Binding ◽  
Naturally Occurring ◽  
Two State Model ◽  
T State

The role of the amino acid at position α38 in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position α38 (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln38 of the human embryonic haemoglobin ζ-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of αGln38 and βTyr145 is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the ζ-chain containing human embryonic haemoglobins.

scholarly journals Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

1999 ◽  
Vol 73 (3) ◽  
pp. 2263-2269 ◽  
Author(s):  
Pascal Cherpillod ◽  
Karin Beck ◽  
Andreas Zurbriggen ◽  
Riccardo Wittek
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Translation Initiation ◽  
Fusion Proteins ◽  
Canine Distemper Virus ◽  
Protein A ◽  
Biological Properties ◽  
Canine Distemper ◽  
Wild Type ◽  
Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans

1991 ◽  
Vol 99 (4) ◽  
pp. 711-719
Author(s):  
K.L. O'Donnell ◽  
A.H. Osmani ◽  
S.A. Osmani ◽  
N.R. Morris
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Essential Gene ◽  
Tetratricopeptide Repeat ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reading Frame ◽  
Integrative Transformation

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 918-924 ◽  
Author(s):  
Eileen Collins Tozer ◽  
Elizabeth K. Baker ◽  
Mark H. Ginsberg ◽  
Joseph C. Loftus
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Single Amino Acid ◽  
Chimeric Receptor ◽  
Amino Acid Residues ◽  
Genetic Approach ◽  
Specific Amino Acid ◽  
Transfected Cells ◽  
Binding Function ◽  
A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

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