scholarly journals Mapping of Effector Binding Sites of Transducin α-Subunit Using Gαt/GαilChimeras

1996 ◽  
Vol 271 (1) ◽  
pp. 413-424 ◽  
Author(s):  
Nikolai P. Skiba ◽  
Hyunsu Bae ◽  
Heidi E. Hamm
Keyword(s):  
Binding Sites ◽  
Α Subunit ◽  
Effector Binding

scholarly journals Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

2000 ◽  
Vol 275 (42) ◽  
pp. 33021-33026 ◽  
Author(s):  
Peter Reichard ◽  
Rolf Eliasson ◽  
Rolf Ingemarson ◽  
Lars Thelander
Keyword(s):  
Cross Talk ◽  
Ribonucleotide Reductase ◽  
Binding Sites ◽  
Allosteric Effector ◽  
Effector Binding

scholarly journals Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tiffany Wu ◽  
Liskin Swint-Kruse ◽  
Aron W. Fenton
Keyword(s):  
Binding Sites ◽  
Functional Outcomes ◽  
Human Liver ◽  
Full Range ◽  
Allosteric Coupling ◽  
Multiple Parameters ◽  
Wide Range ◽  
Allosteric Binding Sites ◽  
Fructose 1,6 Bisphosphate ◽  
Effector Binding

AbstractFor protein mutagenesis, a common expectation is that important positions will behave like on/off “toggle” switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: “rheostat” positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostatic effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of “local” positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostatic outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

scholarly journals Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

2006 ◽  
Vol 37 (2) ◽  
pp. 341-352 ◽  
Author(s):  
Takanobu Sato ◽  
Kousuke Kitahara ◽  
Takao Susa ◽  
Takako Kato ◽  
Yukio Kato
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Gh3 Cells ◽  
Pituitary Hormone ◽  
Transcriptional Level ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone β subunit (FSHβ) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone α subunit (αGSU), and luteinizing hormone β subunit (LHβ). A series of deletion mutants of the porcine αGSU (up to −1059 bp) and LHβ (up to −1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine αGSU and LHβ promoters by Prop-1, which was found to activate the αGSU promoter of −1059/+12 bp up to 11.7-fold but not the LHβ promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, −1038/−1026, −942/−928, −495/−479, −338/−326, −153/−146, and −131/−124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of αGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for αGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of αGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHβ gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of αGSU and FSHβ gene expressions.

scholarly journals Analysis of Allosteric Effector Binding Sites of Potato ADP-glucose Pyrophosphorylase through Reverse Genetics

2001 ◽  
Vol 276 (44) ◽  
pp. 40834-40840 ◽  
Author(s):  
I. Halil Kavakli ◽  
Jong-Sug Park ◽  
Casey J. Slattery ◽  
Peter R. Salamone ◽  
Jennifer Frohlick ◽  
...  
Keyword(s):  
Binding Sites ◽  
Reverse Genetics ◽  
Allosteric Effector ◽  
Adp Glucose Pyrophosphorylase ◽  
Effector Binding

scholarly journals Interleukin-2 (IL-2) Regulates the Accessibility of the IL-2-Responsive Enhancer in the IL-2 Receptor α Gene to Transcription Factors

1999 ◽  
Vol 19 (4) ◽  
pp. 2681-2689 ◽  
Author(s):  
Corinne Rusterholz ◽  
Patricia Corthésy Henrioud ◽  
Markus Nabholz
Keyword(s):  
Transcription Factors ◽  
T Lymphocytes ◽  
Binding Sites ◽  
Interleukin 2 ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Cooperative Binding ◽  
Α Subunit ◽  
Hypersensitive Sites

ABSTRACT Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) α subunit by antigen and by IL-2 itself. IL-2 induces IL-2Rα transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2Rα expression. In cells induced to transiently express IL-2Rα with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2Rα transcription by making IL-2Rα chromatin accessible to transcription factors.

scholarly journals eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

2020 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

scholarly journals eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

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