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Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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crystal structure and biochemical characterization of a mycobacterium smegmatis aaa type nucleoside triphosphatase phosphohydrolase msm0858
Journal of Bacteriology, 2016Co-Authors: Mihaelacarmen Unciuleac, Paul Smith, Stewart ShumanAbstract:ABSTRACT AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-A crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. IMPORTANCE Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.
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mutational analysis of baculovirus capping enzyme lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity
Journal of Biological Chemistry, 2001Co-Authors: Alexandra Martins, Stewart ShumanAbstract:Abstract The 464-amino acid baculovirus Lef4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The hydrolysis of 5′-triphosphate RNA and free NTPs by Lef4 is dependent on a divalent cation cofactor. RNA triphosphatase activity is optimal at pH 7.5 with either magnesium or manganese, yet NTP hydrolysis at neutral pH is activated only by manganese or cobalt. Here we show that Lef4 possesses an intrinsic magnesium-dependent ATPase with a distinctive alkaline pH optimum and a high K m for ATP (4 mm). Lef4 contains two conserved sequences, motif A (8IEKEISY14) and motif C (180LEYEF184), which define the fungal/viral/protozoal family of metal-dependent RNA triphosphatases. We find by mutational analysis that Glu9, Glu11, Glu181, and Glu183 are essential for phosphohydrolase chemistry and likely comprise the metal-binding site of Lef4. Conservative mutations E9D and E183D abrogate the magnesium-dependent triphosphatase activities of Lef4 and transform it into a strictly manganese-dependent RNA triphosphatase. Limited proteolysis of Lef4 and ensuing COOH-terminal deletion analysis revealed that the NH2-terminal 236-amino acid segment of Lef4 constitutes an autonomous triphosphatase catalytic domain.
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YEAST AND VIRAL RNA 5' TRIPHOSPHATASES COMPRISE A NEW NUCLEOSIDE TRIPHOSPHATASE FAMILY
The Journal of biological chemistry, 1998Co-Authors: Yi Pei, Stewart ShumanAbstract:Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, the hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium-dependent and has a turnover number of 1 s-1. Here we show that purified recombinant Cet1p possesses a robust ATPase activity (Km = 2.8 microM; Vmax = 25 s-1) in the presence of manganese. Cobalt is also an effective cofactor, but magnesium, calcium, copper, and zinc are not. Cet1p displays broad specificity in converting ribonucleoside triphosphates and deoxynucleoside triphosphates to their respective diphosphates. The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs. Mutational analysis establishes that conserved glutamate and arginine side chains within these motifs are essential for the RNA triphosphatase and ATPase activities of Cet1p in vitro and for Cet1p function in vivo. These findings are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel family of phosphohydrolase enzymes with a common active site.
David A. Agard - One of the best experts on this subject based on the ideXlab platform.
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calcium binding to a remote site can replace magnesium as cofactor for mitochondrial hsp90 trap1 atpase activity
Journal of Biological Chemistry, 2018Co-Authors: Daniel Elnatan, David A. AgardAbstract:The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor–associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients.
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modulation of mitochondrial hsp90 trap1 atpase activity by calcium and magnesium
bioRxiv, 2018Co-Authors: David A. Agard, Daniel ElnatanAbstract:The Hsp90 protein family are ATP-dependent molecular chaperones that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotic cells have organelle-specific Hsp90 paralogs that are adapted to each unique sub-cellular environment. The mitochondrial Hsp90, TRAP1, supports the folding and activity of electron transport components and is increasingly being appreciated as a critical player in mitochondrial signaling. It is well known that calcium plays an important regulatory role in mitochondria and can even accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we find that calcium can replace the requirement for magnesium to support TRAP1 ATPase activity. Using anomalous x-ray diffraction, we reveal a novel calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP γ-phosphate and completely distinct from the magnesium site adjacent to the α- and β-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, is non-cooperative, whereas calcium binding results in cooperative ATP hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggest a mechanism for the cooperative behavior. Owing to the cooperativity, at high ATP concentrations, ATPase activity is higher with calcium, whereas the converse is true at low ATP concentrations. Integrating these observations, we propose a model where the divalent cations choice can control switching between non-cooperative and cooperative TRAP1 ATPase mechanisms in response ATP concentrations. This may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle.
Daniel Elnatan - One of the best experts on this subject based on the ideXlab platform.
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calcium binding to a remote site can replace magnesium as cofactor for mitochondrial hsp90 trap1 atpase activity
Journal of Biological Chemistry, 2018Co-Authors: Daniel Elnatan, David A. AgardAbstract:The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor–associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients.
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modulation of mitochondrial hsp90 trap1 atpase activity by calcium and magnesium
bioRxiv, 2018Co-Authors: David A. Agard, Daniel ElnatanAbstract:The Hsp90 protein family are ATP-dependent molecular chaperones that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotic cells have organelle-specific Hsp90 paralogs that are adapted to each unique sub-cellular environment. The mitochondrial Hsp90, TRAP1, supports the folding and activity of electron transport components and is increasingly being appreciated as a critical player in mitochondrial signaling. It is well known that calcium plays an important regulatory role in mitochondria and can even accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we find that calcium can replace the requirement for magnesium to support TRAP1 ATPase activity. Using anomalous x-ray diffraction, we reveal a novel calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP γ-phosphate and completely distinct from the magnesium site adjacent to the α- and β-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, is non-cooperative, whereas calcium binding results in cooperative ATP hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggest a mechanism for the cooperative behavior. Owing to the cooperativity, at high ATP concentrations, ATPase activity is higher with calcium, whereas the converse is true at low ATP concentrations. Integrating these observations, we propose a model where the divalent cations choice can control switching between non-cooperative and cooperative TRAP1 ATPase mechanisms in response ATP concentrations. This may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle.
Lee H Sweeney - One of the best experts on this subject based on the ideXlab platform.
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Magnesium Regulates ADP Dissociation From Myosin V
2013Co-Authors: Steven S. Rosenfeld, Anne Houdusse, Lee H SweeneyAbstract:Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release, and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V:MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin in order to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor
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magnesium regulates adp dissociation from myosin v
Journal of Biological Chemistry, 2005Co-Authors: Steven S. Rosenfeld, Anne Houdusse, Lee H SweeneyAbstract:Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.
Ildan F. - One of the best experts on this subject based on the ideXlab platform.
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The effect of the treatment of high-dose methylprednisolone on Na+-K+/Mg+2 ATPase activity and lipid peroxidation and ultrastructural findings following cerebral contusion in rat
ELSEVIER SCIENCE INC, 1995Co-Authors: Ildan F., Polat S., Kaya M., Öner A., Isbir T., Cetinalp E, Karadayi AAbstract:WOS: A1995TX87100028PubMed ID: 8669035BACKGROUND Although use of corticosteroid in the management of head trauma has caused a great deal of controversy, corticosteroids have long been an adjunct in the management of severe closed head injury. The glucocorticoid steroid methylprednisolone (MP) has been proven to have significant antioxidant effect when administered in an antioxidant-high dose after central nervous system injury. METHODS The sodium-potassium activated and magnesium dependent adenosine-5'-triphosphatase (Na+-K+/Mg+2 ATPase EC.3.6.1.3.) activity, lipid peroxidation, and early ultrastructural findings were determined during the immediate posttraumatic period in rats. Mechanical brain injury was produced when a calibrated weight-drop device is allowed to fall on to the skull's convexity over the right hemisphere, 1 to 2 mm lateral from the midline. In group I, rats were used to determine Na+-K+/Mg+2 ATPase activity, the extent of lipid peroxidation, by measuring the level of malondialdehyde content and normal ultrastructural findings in two different brain areas (cerebral cortex and brain stem), In group II, physiologic saline was administered right after trauma in the same amount as methylprednisolone. In group III rats, methylprednisolone (30 mg/kg) was administered intravenously right after trauma. RESULTS Na+-K+/Mg+2 ATPase activity significantly decreased in the cerebral cortex and in brain stem within 2 hours after trauma (p < 0.05), There was significant difference in malondialdehyde content between groups II and III (p < 0.05). Methylprednisolone treatment reduced malondialdehyde content and induced the recovery of Na+-K+/Mg+2 activity. CONCLUSIONS These data suggest that inactivation of Na+-K+/Mg+2 ATPase is closely correlated to changes of lipid peroxidation and the alteration of the ultrastructural findings in the early phases after head trauma, The glucocorticoid steroid methylprednisolone has been proven to have significant effect in inactivation of Na+-K+/Mg+2 ATPase with significant reduction of malondialdehyde content
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Correlation of alterations on Na+-K+/Mg+2 ATPase activity, lipid peroxidation and ultrastructural findings following experimental spinal cord injury with and without intravenous methylprednisolone treatment
'Springer Science and Business Media LLC', 1995Co-Authors: Ildan F., Polat S., Gocer A.i., Kaya M., Öner A., Isbir T., Karaday A.Abstract:PubMedID: 7566528The sodium-potassium activated and magnesium dependent adenosine-5'-triphosphatase (Na+-K+/Mg+2 ATPase EC 3.6.1.3.) activity and lipid peroxidation and early ultrastructural findings are determined in rat spinal cord at the early stage of trauma produced by a surgical clip on the thoracal 2-7 segments. The effect of treatment with intravenous methylprednisolone (MP) was evaluated the basis of these biochemical alterations and ultrastructural findings in the same model. The specific activity of the membrane bound enzyme Na+-K+/Mg+2 ATPase was promptly reduced in as early as ten minutes following spinal cord injury and remained at a level lower than the levels in the control group and in the sham-operated group. Methylprednisolone treatment immediately after the trauma attenuated the inactivation of Na+-K+/Mg+2 ATPase. On the other hand, there was significant difference in lipid peroxide content between the sham-operated and the injured animals. Methylprednisolone treatment reduced thiobarbituric acid reactive substance (TBARS) content in Group IV. We determined a positive relationship among membrane-bound enzyme Na+ K+/Mg+2 ATPase activity, malondialdehyde (MDA) content and early ultrastructural changes in the traumatized and treated groups. These data provide evidence for a beneficial effect of methylprednisolone on the activation of Na+-K+/Mg+2 ATPase and lipid peroxidation and early ultrastructural changes in spinal cord injured rats. The possible mechanism of methylprednisolone effects on the membrane function and lipid peroxidation, and the correlation of biochemical changes with ultrastructural findings are briefly discussed. © 1995 Walter de Gruyter & Co
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CORRELATION OF ALTERATIONS ON NA+-K+MG+2 ATPASE ACTIVITY, LIPID-PEROXIDATION AND ULTRASTRUCTURAL FINDINGS FOLLOWING EXPERIMENTAL SPINAL-CORD INJURY WITH AND WITHOUT INTRAVENOUS METHYLPREDNISOLONE TREATMENT
'Springer Science and Business Media LLC', 1995Co-Authors: Ildan F., Polat S., Kaya M., Öner A., Isbir T., Ai Gocer, Karadayi AAbstract:WOS: A1995RG37300006PubMed ID: 7566528The sodium-potassium activated and magnesium dependent adenosine-5'-triphosphatase (Na+-K+/Mg+2 AT- Pase EC 3.6.1.3.) activity and lipid peroxidation and early ultrastructural findings are determined in rat spinal cord at the early stage of trauma produced by a surgical clip on the thoracal 2-7 segments. The effect of treatment with intravenous methylprednisolone (MP) was evaluated the basis of these biochemical alterations and ultrastructural findings in the same model. The specific activity of the membrane bound enzyme Na+-K+/Mg+2 ATPase was promptly reduced in as early as ten minutes following spinal cord injury and remained at a level lower than the levels in the control group and in the sham-operated group. Methylprednisolone treatment immediately after the trauma attenuated the inactivation of Na+-K+/Mg+2 ATPase. On the other hand, there was significant difference in lipid peroxide content between the sham-operated and the injured animals. Methylprednisolone treatment reduced thiobarbituric acid reactive substance (TEARS) content in Group IV. We determined a positive relationship among membrane-bound enzyme Na+K+/Mg+2 ATPase activity, malondialdehyde (MDA) content and early ultrastructural changes in the traumatized and treated groups. These data provide evidence for a beneficial effect of methylprednisolone on the activation of Na+-K+/Mg+2 ATPase and lipid peroxidation and early ultrastructural changes in spinal cord injured rats. The possible mechanism of methylprednisolone effects on the membrane function and lipid peroxidation, and the correlation of biochemical changes with ultrastructural findings are briefly discussed
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The effect of thyrotropin releasing hormone (TRH) on the experimental carbon dioxide laser brain lesion: Ultrastructural and biochemical study
'Turkish Neurosurgical Society', 1994Co-Authors: Bagdatoglu H., Ildan F., Polat S., Gocer A.i., Herguner K., Aksoy K., Kaya M.Abstract:Although the main advantage of the CO2 laser lies in the possibility of a less traumatic effect on the surrounding tissue, its use in neurosurgery still necessitates a thorough and detailed evaluation of the effect on surrounding normal central nervous system (CNS) tissue, Therefore this study was undertaken to investigate the ultrastructural and biochemical effects of the CO2 laser on the application area and the surrounding normal central nervous system tissue. Sodium-potassium activated and magnesium-dependent adenosine-5'-triphosphatase (Na+-K+/Mg2+ ATPase E.C.3.6.3.1), magnesium dependent adenosine-5'-triphosphatase (Mg2+ ATPase E.C.3.6.1.3) and calcium activated magnesium dependent adenosine-5'-triphosphatase (Ca2+/Mg2+ ATPase E.C.3.6.1.3) enzymes, superoxide dismutase, light microscopic and ultrastructural findings were determined in dog brain following laser application with and without thyrotropin releasing hormone (TRH) treatment. Laser lesions were created by a CO2 laser in the cerebrum. Fifteen days later, after thyrotropin releasing hormone injection, ultrastructural and biochemical investigations were undertaken to evaluate the effect of thyrotropin releasing hormone on the laser induced lesion and particularly surrounding cerebral tissue. Ultrastructural findings, showed that thyrotropin releasing hormone reduced degeneration on the CO2 laser-applied lesion
