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Alan M Lambowitz - One of the best experts on this subject based on the ideXlab platform.
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function of tyrosyl trna synthetase in splicing group i introns an Induced Fit Model for binding to the p4 p6 domain based on analysis of mutations at the junction of the p4 p6 stacked helices
Journal of Molecular Biology, 2000Co-Authors: Xin Chen, Robin R Gutell, Alan M LambowitzAbstract:Abstract We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants. Most mutations at either position inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking, based on the analysis of DNA four-way junctions. However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressible mutants bind the protein with K d values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k off value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K d value reflects primarily an increased k on value for the binding of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core.
Anthony P H Wright - One of the best experts on this subject based on the ideXlab platform.
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functional interaction of the c myc transactivation domain with the tata binding protein evidence for an Induced Fit Model of transactivation domain folding
Biochemistry, 1996Co-Authors: Iain J Mcewan, Karin Dahlmanwright, Jacqueline Ford, Anthony P H WrightAbstract:c-Myc is a member of a family of sequence specific-DNA binding proteins that are thought to regulate the transcription of genes involved in normal cell growth, differentiation, and apoptosis. In order to understand how human c-myc functions as a transcription factor, we have studied the mechanism of action and structure of the N-terminal transactivation domain, amino acids 1-143. In a protein interaction assay, c-myc1-143 bound selectively to two basal transcription factors, the TATA binding protein (TBP) and the RAP74 subunit of TFIIF. Furthermore, the isolated c-myc transactivation domain competed for limiting factors required for the assembly of a functional preinitiation complex. This squelching of basal transcription was reversed in a dose-dependent manner by recombinant TBP. Taken together, these results identify TBP as an important target for the c-myc transactivation domain, during transcriptional initiation. Similar to other transactivation domains, the c-myc1-143 polypeptide showed little or no evidence of secondary structure, when measured by circular dichroism spectroscopy (CD) in aqueous solution. However, significant alpha-helical conformation was observed in the presence of the hydrophobic solvent trifluoroethanol. Strikingly, addition of TBP caused changes in the CD spectra consistent with induction of protein conformation in c-myc1-143 during interaction with the target factor. This change was specific for TBP as a similar effect was not observed in the presence of TFIIB. These data support a Model in which target factors induce or stabilize a structural conformation in activator proteins during transcriptional transactivation.
Touzé Thierry - One of the best experts on this subject based on the ideXlab platform.
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Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.
2020Co-Authors: Tian Xudong, Auger Rodolphe, Manat Guillaume, Kerff Frédéric, Mengin-lecreulx Dominique, Touzé ThierryAbstract:Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C(55)-P). C(55)-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C(55)-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C(55)-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an Induced-Fit Model of action.Peer reviewe
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Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli
'Springer Science and Business Media LLC', 2020Co-Authors: Tian Xudong, Auger Rodolphe, Manat Guillaume, Kerff Frédéric, Mengin-lecreulx Dominique, Touzé ThierryAbstract:International audienceUbiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an Induced-Fit Model of action
Xin Chen - One of the best experts on this subject based on the ideXlab platform.
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function of tyrosyl trna synthetase in splicing group i introns an Induced Fit Model for binding to the p4 p6 domain based on analysis of mutations at the junction of the p4 p6 stacked helices
Journal of Molecular Biology, 2000Co-Authors: Xin Chen, Robin R Gutell, Alan M LambowitzAbstract:Abstract We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants. Most mutations at either position inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking, based on the analysis of DNA four-way junctions. However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressible mutants bind the protein with K d values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k off value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K d value reflects primarily an increased k on value for the binding of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core.
Vlatko Vedral - One of the best experts on this subject based on the ideXlab platform.
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proton tunnelling in hydrogen bonds and its implications in an Induced Fit Model of enzyme catalysis
Proceedings of The Royal Society A: Mathematical Physical and Engineering Sciences, 2018Co-Authors: Onur Pusuluk, Tristan Farrow, Cemsinan Deliduman, Keith Burnett, Vlatko VedralAbstract:The role of proton tunnelling in biological catalysis is investigated here within the frameworks of quantum information theory and thermodynamics. We consider the quantum correlations generated through two hydrogen bonds between a substrate and a prototypical enzyme that first catalyses the tautomerization of the substrate to move on to a subsequent catalysis, and discuss how the enzyme can derive its catalytic potency from these correlations. In particular, we show that classical changes Induced in the binding site of the enzyme spreads the quantum correlations among all of the four hydrogen-bonded atoms thanks to the directionality of hydrogen bonds. If the enzyme rapidly returns to its initial state after the binding stage, the substrate ends in a new transition state corresponding to a quantum superposition. Open quantum system dynamics can then naturally drive the reaction in the forward direction from the major tautomeric form to the minor tautomeric form without needing any additional catalytic activity. We find that in this scenario the enzyme lowers the activation energy so much that there is no energy barrier left in the tautomerization, even if the quantum correlations quickly decay.