scholarly journals Structural Basis for Actin Assembly, Activation of ATP Hydrolysis, and Delayed Phosphate Release

Cell ◽  
2010 ◽  
Vol 143 (2) ◽  
pp. 275-287 ◽  
Author(s):  
Kenji Murakami ◽  
Takuo Yasunaga ◽  
Taro Q.P. Noguchi ◽  
Yuki Gomibuchi ◽  
Kien X. Ngo ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Structural Basis ◽  
Actin Assembly ◽  
Phosphate Release

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

scholarly journals Structural basis for DEAH-helicase activation by G-patch proteins

2020 ◽  
Vol 117 (13) ◽  
pp. 7159-7170 ◽  
Author(s):  
Michael K. Studer ◽  
Lazar Ivanović ◽  
Marco E. Weber ◽  
Sabrina Marti ◽  
Stefanie Jonas
Keyword(s):  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Rna Helicases ◽  
Catalytic Core ◽  
Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

scholarly journals Polymerization of actin by positively charged liposomes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1221-1227 ◽  
Author(s):  
A Laliberte ◽  
C Gicquaud
Keyword(s):  
Electron Microscopy ◽  
Direct Contact ◽  
Atp Hydrolysis ◽  
Actin Assembly ◽  
Liposome Membrane ◽  
Low Salt ◽  
Spectrofluorimetric Analysis ◽  
Positively Charged

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.

The Elegant Platelet: Signals Controlling Actin Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 392-398 ◽  
Author(s):  
Kurt Barkalow ◽  
Anser Azim ◽  
Joe Italiano ◽  
John Hartwig
Keyword(s):  
Actin Filaments ◽  
Active Form ◽  
Blood Platelet ◽  
Vascular Damage ◽  
Structural Basis ◽  
Actin Assembly ◽  
Small Disc ◽  
Specific Proteins

SummaryThe function of the blood platelet is defined by its two physical states. The platelet is born in the first state, in long cytoplasmic extensions from megakaryocytes, as a small disc of reproducible structure having an elegant, actin-based cytoskeleton. At rest, the platelet circulates through the vasculature in this form. In the second state, in response to vascular damage, the platelet rapidly converts into its active form with filopodia and lamellipodia that derive from a remodeled actin skeleton and a massive assembly of new actin filaments. In the last two decades, we have begun to understand the structural basis for these two platelet states, the role of specific proteins that define the architecture of both shapes, and the proteins and signals that drive this conversion of shapes.

G-actin conformational change and polymerization induced by paraquat

1998 ◽  
Vol 76 (4) ◽  
pp. 583-591 ◽  
Author(s):  
Isabella DalleDonne ◽  
Aldo Milzani ◽  
Roberto Colombo
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Cell Injury ◽  
Atp Hydrolysis ◽  
Protein Composition ◽  
Dnase I ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Actin Assembly

Paraquat (1,1´-dimethyl-4,4´-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.Key words: actin, paraquat, subdomain 2, DNase I, ATP hydrolysis.

The Structural Basis for Coupling of Ca2+ Transport to ATP Hydrolysis by the Sarcoplasmic Reticulum Ca2+-ATPase

2005 ◽  
Vol 37 (6) ◽  
pp. 359-364 ◽  
Author(s):  
Jesper Vuust Møller ◽  
Claus Olesen ◽  
Anne-Marie Lund Jensen ◽  
Poul Nissen
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Structural Basis

scholarly journals Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Prattes ◽  
Irina Grishkovskaya ◽  
Victor-Valentin Hodirnau ◽  
Ingrid Rössler ◽  
Isabella Klein ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Atp Hydrolysis ◽  
Ribosomal Subunit ◽  
Covalent Bond ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
Structural Basis ◽  
Aaa Atpase ◽  
Maturation Factor ◽  
Key Factor

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

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