scholarly journals Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle

2018 ◽  
Vol 4 (9) ◽  
pp. eaau4196 ◽  
Author(s):  
Guillaume Mas ◽  
Jia-Ying Guan ◽  
Elodie Crublet ◽  
Elisa Colas Debled ◽  
Christine Moriscot ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Nucleotide Binding ◽  
Ring Structure ◽  
Structural Level ◽  
Dependent Manner ◽  
Regeneration System ◽  
X Ray Crystallography ◽  
Molecular Events ◽  
Client Proteins ◽  
Atp Regeneration System

Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5′-triphosphate (ATP)–dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.

The Pyrolytic Rearrangement of 1-Alkynoyl-3-methylpyrazoles: Synthesis of Pyrazolo[1,5-a]pyridin-5-ols and Related Compounds

1994 ◽  
Vol 47 (6) ◽  
pp. 991 ◽  
Author(s):  
RFC Brown ◽  
FW Eastwood ◽  
GD Fallon ◽  
SC Lee ◽  
RP Mcgeary
Keyword(s):  
Carbon Chain ◽  
Ring Structure ◽  
High Yield ◽  
Methyl Group ◽  
X Ray ◽  
X Ray Crystallography ◽  
Flash Vacuum Pyrolysis ◽  
Vacuum Pyrolysis ◽  
Fused Ring ◽  
Related Compounds

Flash vacuum pyrolysis of 1-(alkyn-2′-oyl)-3-methylpyrazoles at 650°/0.03 mm forms pyrazolo[1,5-a]pyridin-5-ols, often in high yield, which may bear substituents at C2, C3 or C7. In the absence of a 3-methyl group in the precursor, N-ethynylpyrazoles are formed in low yield. The formation of both types of product is interpreted as involving 3-(N-pyrazolyl)propadienones formed by N1 → N2 migration of the N-alkynoyl group with inversion of the three-carbon chain. The fused-ring structure of 2-methylpyrazolo[1,5-a]pyridin-5-ol (25) was established by X-ray crystallography of the O-benzoyl derivative (27).

Gold(I) Complexes Bearing P∩N-Ligands: An Unprecedented Twelve-membered Ring Structure Stabilized by Aurophilic Interactions

2009 ◽  
Vol 64 (11-12) ◽  
pp. 1513-1524 ◽  
Author(s):  
Uwe Monkowius ◽  
Manfred Zabel ◽  
Michel Fleck ◽  
Hartmut Yersin
Keyword(s):  
Solid State ◽  
Ring Structure ◽  
Phosphine Ligands ◽  
X Ray Crystallography ◽  
Aggregation Pattern ◽  
Elemental Analyses ◽  
High Yields ◽  
First Time ◽  
Solid State Structures ◽  
Aurophilic Interactions

The P∩N-ligands Ph2Pqn, 1, Ph2 Piqn, 2, Ph2 Ppym, 3, and the As∩N-ligands Ph2Asqn, 4, Ph2Asiqn, 5, (Ph = phenyl, qn = 8-quinoline, iqn = 1-isoquinoline, pym = 2-pyrimidine) have been synthesized, the ligands 2 and 5 for the first time. Their ligand properties were probed by the synthesis of gold(I) complexes. Reaction with (tht)AuCl (tht = tetrahydrothiophene) yielded the chlorogold complexes Ph2RP-Au-Cl (R = qn, 6; iqn, 7; pym, 8) and Ph2RAs-Au-Cl (R = qn, 9; iqn, 10) in high yields. Further treatment of 7 and 8 with one equivalent of AgBF4 provided the complexes [(Ph2Piqn)Au]BF4, 11, [(Ph2Ppym)Au]BF4, 12, and [(Ph2Piqn)Au(tht)]BF4, 14. For comparison, the previously reported complex [(Ph2Ppy)Au]BF4 (py = pyridine), 13, was re-investigated. The compounds were characterized by elemental analyses, mass spectrometry and NMR spectroscopy. In addition, the solid-state structures of 2, 3, 6, 7, 9 - 14 have been determined by X-ray crystallography. The chloro-gold compounds crystallize in the common rod-like structure known from R3EAuCl (R = aryl, E = P, As) complexes without further aggregation via aurophilic interactions. In all cases the phosphine acts as a monodentate ligand. In the solid state compounds 11 - 13 feature an unprecedented cyclic trinuclear aggregation pattern, in which the Au(I) atoms are linearly coordinated by the bridging phosphine ligands forming a cyclic (P-Au-N)3 arrangement. The resulting twelvemembered ring is further stabilized by Au · · · Au interactions. Due to the presence of these Au · · · Au contacts, 11 - 13 are emissive in the solid state but not in solution

scholarly journals Role of the HSP70 Co-Chaperone SIL1 in Health and Disease

2021 ◽  
Vol 22 (4) ◽  
pp. 1564
Author(s):  
Viraj P. Ichhaporia ◽  
Linda M. Hendershot
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Multisystem Disorder ◽  
Nucleotide Exchange Factors ◽  
Precise Understanding ◽  
Health And Disease ◽  
Client Proteins ◽  
Er Homeostasis

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.

scholarly journals Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

1999 ◽  
Vol 46 (2) ◽  
pp. 419-429 ◽  
Author(s):  
M Danieluk ◽  
R Buś ◽  
S Pikuła ◽  
J Bandorowicz-Pikuła
Keyword(s):  
Binding Site ◽  
Binding Proteins ◽  
Direct Sequencing ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Dependent Manner ◽  
Cibacron Blue ◽  
Annexin Vi ◽  
Cibacron Blue 3Ga ◽  
Triazine Dye

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.

scholarly journals High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing

2018 ◽  
Vol 115 (6) ◽  
pp. 1292-1297 ◽  
Author(s):  
Ahmet Mentes ◽  
Andrew Huehn ◽  
Xueqi Liu ◽  
Adam Zwolak ◽  
Roberto Dominguez ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Bound States ◽  
Nucleotide Binding ◽  
Structural Basis ◽  
Force Sensing ◽  
Dependent Manner ◽  
Mechanical Loads ◽  
Adp Release ◽  
Pointed End

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP

1998 ◽  
Vol 111 (24) ◽  
pp. 3675-3686 ◽  
Author(s):  
D. Lourim ◽  
G. Krohne
Keyword(s):  
Xenopus Laevis Oocytes ◽  
Chromatin Binding ◽  
Regeneration System ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg ◽  
Atp Regeneration System ◽  
Opposing Effects ◽  
Xenopus Egg Extracts ◽  
Lambda Dna

We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms.

scholarly journals Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies)

Mycorrhiza ◽  
2020 ◽  
Author(s):  
Oluwatosin Abdulsalam ◽  
Katharina Wagner ◽  
Sophia Wirth ◽  
Maritta Kunert ◽  
Anja David ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Volatile Organic Compounds ◽  
Picea Abies ◽  
Norway Spruce ◽  
Organic Compounds ◽  
Mycorrhizal Fungi ◽  
Isotope Labeling ◽  
Dependent Manner ◽  
Hyphal Branching ◽  
Volatile Organic

AbstractThe ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and β-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

Cyclic AMP-dependent protein kinase enhances hippocampal dentate granule cell GABAA receptor currents

1996 ◽  
Vol 76 (4) ◽  
pp. 2626-2634 ◽  
Author(s):  
J. Kapur ◽  
R. L. Macdonald
Keyword(s):  
Protein Kinase ◽  
Granule Cells ◽  
Regeneration System ◽  
Gaba Concentration ◽  
Dentate Granule Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
The Mean ◽  
Atp Regeneration System ◽  
Atp Regeneration

1. The effects of activation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), intracellular application of PKA and inhibition of endogenous PKA by protein kinase inhibitory peptide (PKIP) on hippocampal dentate granule cell gamma-aminobuturic acid A (GABAA) receptor (GABAR) currents were characterized. 2. GABAR currents evoked by repeated application of GABA (30 or 100 microM) were enhanced by application of 1 mM norepinephrine (52 +/- 26%; mean +/- SE; n = 11) and of 500 mM 8-bromo cAMP (15 +/- 2%, n = 7). 3. GABA concentration response curves were obtained from six dentate granule cells before and after application of 500 microM 8-bromo cAMP. The maximal current was increased significantly by 89 +/- 36%, but the mean EC50 was not significantly changed (68 +/- 42 microM vs. 25 +/- 10 microM). 4. The GABA concentration response relationship was studied in a group of 7 granule cells recorded with pipettes containing PKIP and 2 mM ATP and compared with another group of 12 cells recorded with 2 mM ATP in the pipette. When currents were recorded with intracellular PKIP, the mean EC50 for GABA was no different (43 +/- 9 microM vs. 45 +/- 16 microM); however, the maximal current obtained was smaller, (961 +/- 102 pA vs. 658 +/- 104 pA). 5. Concentration response data were obtained from four granule cells using recording pipettes containing the cPKA and an ATP regeneration system and compared with seven cells recorded with the ATP regeneration system. With cPKA, the maximal GABAR current was significantly larger (1,224 +/- 132 pA vs. 718 +/- 56 pA), but the EC50 for GABA was not significantly altered (21 +/- 2.0 microM vs. 79 +/- 25 microM).

scholarly journals Listeria monocytogenesActivated p38 MAPK and Induced IL-8 Secretion in a Nucleotide-Binding Oligomerization Domain 1-Dependent Manner in Endothelial Cells

2005 ◽  
Vol 176 (1) ◽  
pp. 484-490 ◽  
Author(s):  
Bastian Opitz ◽  
Anja Püschel ◽  
Wiebke Beermann ◽  
Andreas C. Hocke ◽  
Stefanie Förster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
P38 Mapk ◽  
Nucleotide Binding ◽  
Dependent Manner ◽  
Oligomerization Domain
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