scholarly journals Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

2014 ◽  
Vol 70 (2) ◽  
pp. 582-595 ◽  
Author(s):  
Cathleen Zeymer ◽  
Thomas R. M. Barends ◽  
Nicolas D. Werbeck ◽  
Ilme Schlichting ◽  
Jochen Reinstein
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Molecular Motor ◽  
Nucleotide Binding ◽  
Side Chain ◽  
Side Chain Conformation ◽  
Proposed Model ◽  
Hydrolysis Of ◽  
Conserved Residue

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg2+ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

scholarly journals “Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

2015 ◽  
Vol 1 (7) ◽  
pp. e1500263 ◽  
Author(s):  
Akihiko Nakamura ◽  
Takuya Ishida ◽  
Katsuhiro Kusaka ◽  
Taro Yamada ◽  
Shinya Fushinobu ◽  
...  
Keyword(s):  
Glycoside Hydrolases ◽  
Side Chain ◽  
Enzymatic Reactions ◽  
X Ray Diffraction ◽  
Peptide Bonds ◽  
Proton Relay ◽  
Newton’S Cradle ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

scholarly journals The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1970 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
P. S. G. Goldfarb ◽  
R. Rodnight
Keyword(s):  
Potassium Chloride ◽  
Magnesium Chloride ◽  
Adenosine Triphosphatase ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Protein Ratio ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

Tubulin folding is altered by mutations in a putative GTP binding motif

1996 ◽  
Vol 109 (6) ◽  
pp. 1471-1478 ◽  
Author(s):  
J.C. Zabala ◽  
A. Fontalba ◽  
J. Avila
Keyword(s):  
Intramolecular Interaction ◽  
Terminal Region ◽  
Binding Motif ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Proposed Model ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hydrolysis Of

Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An alpha-tubulin point mutant with a T150--&gt;G substitution (the corresponding residue present in beta-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four beta-tubulin point mutants, including the G148--&gt;T substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (intermediates in the process of tubulin folding). Three other mutants within this region (which approximately encompasses residues 137–152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

2001 ◽  
Vol 21 (2) ◽  
pp. 113-137 ◽  
Author(s):  
Leopoldo de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Surrounding Medium ◽  
Chemical Energy ◽  
The Past ◽  
Sarcoplasmic Reticulum Membrane ◽  
Membrane Bound ◽  
Hydrolysis Of ◽  
Heat Released

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

scholarly journals Intracellular Proton-Transfer Mutants in a CLC Cl−/H+ Exchanger

2009 ◽  
Vol 133 (2) ◽  
pp. 131-138 ◽  
Author(s):  
Hyun-Ho Lim ◽  
Christopher Miller
Keyword(s):  
Proton Transfer ◽  
Mutational Analysis ◽  
Side Chain ◽  
Side Chains ◽  
Extracellular Water ◽  
Wild Type ◽  
Cl Transport ◽  
Structural And Functional Properties ◽  
Key Residues

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl− and H+. Mutational analysis based on the known structure reveals several key residues required for coupling H+ to the stoichiometric countermovement of Cl−. E148 (Gluex) transfers protons between extracellular water and the protein interior, and E203 (Gluin) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H+ transport while preserving Cl− transport. We tested the role of Gluin by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H+/Cl− exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H+ coupling and severely inhibit Cl− flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H+ transport with substantial Cl− transport. Influence on H+ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Gluin is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl− movements to bring about Cl−/H+ exchange.

The role of ATP in the reactions of type II DNA topoisomerases

2010 ◽  
Vol 38 (2) ◽  
pp. 438-442 ◽  
Author(s):  
Andrew D. Bates ◽  
Anthony Maxwell
Keyword(s):  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Topology ◽  
Type Ii ◽  
Free Energies ◽  
Dna Topoisomerases ◽  
Type Ii Dna Topoisomerases ◽  
Topology Simplification ◽  
Hydrolysis Of

Type II DNA topoisomerases catalyse changes in DNA topology in reactions coupled to the hydrolysis of ATP. In the case of DNA gyrase, which can introduce supercoils into DNA, the requirement for free energy is clear. However, the non-supercoiling type II enzymes carry out reactions that are apparently energetically favourable, so their requirement for ATP hydrolysis is not so obvious. It has been shown that many of these enzymes (the type IIA family) can simplify the topology of their DNA substrates to a level beyond that expected at equilibrium. Although this seems to explain their usage of ATP, we show that the free energies involved in topology simplification are very small (<0.2% of that available from ATP) and we argue that topology simplification may simply be an evolutionary relic.

Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation

2011 ◽  
Vol 18 (1) ◽  
pp. 100-111 ◽  
Author(s):  
Birte Zurek ◽  
Martina Proell ◽  
Roland N Wagner ◽  
Robert Schwarzenbacher ◽  
Thomas A Kufer
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Inflammatory Diseases ◽  
Nucleotide Binding ◽  
Blau Syndrome ◽  
Innate Immune Responses ◽  
Atpase Domain ◽  
Early Onset Sarcoidosis ◽  
Key Residues ◽  
Oligomerization Domain

Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.

scholarly journals Visualizing ATP hydrolysis in a viral DNA-packaging molecular motor

2014 ◽  
Vol 70 (a1) ◽  
pp. C1604-C1604
Author(s):  
Liang Tang ◽  
Haiyan Zhao ◽  
Theodore Christensen ◽  
Zihan Lin ◽  
Annie Lynn
Keyword(s):  
Chemical Reaction ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Translational Motion ◽  
Alpha Helix ◽  
Dna Packaging ◽  
Side Chain ◽  
Viral Dna ◽  
Viral Dna Packaging

Many DNA viruses encode powerful molecular machines to package viral genome into preformed protein shells. These DNA-packaging motors contain an ATPase module that converts the chemical reaction of ATP hydrolysis to physical motion of DNA. We previously determined the structures of the DNA-packaging motor gp2 of Shigella phage Sf6 in the apo form and in complex with ADP and ATP-gamma-S (Zhao et al, 2013, PNAS, 110, 8075). Here we report the structure of gp2 in complex with its substrate ATP at 2.0 Angstrom resolution. To our knowledge, this is the first time to capture, at high resolution, a precatalytic state for ASCE-superfamily ATPases, which include a large group of nucleic acid helicases and translocases involved in a broad range of cellular and viral processes. The structure reveals the precise architecture of the ATP-bound state of the motor immediately prior to catalysis. Comparison with structures of the apo and ADP-complexed forms unveils motions of the Walker A motif coupled with ATP and Mg2+ binding and ATP hydrolysis. In the Walker B motif, residue E118 undergoes a side chain conformational switching coupled with the ATP hydrolysis, whereas residue E119 locks residue R51 side chain to a conformation that is readily reachable to residue E118 side chain. Residue E121 in the Walker B motif deprotonates a water molecule, which acts as a nucleophile to attack the gamma-phosphorous, leading to ATP hydrolysis. The alpha-helix (residue G182-R194) in the linker domain undergoes a translational motion against the ATPase domain triggered by ATP hydrolysis, serving as a mechanism for translating the energy from the chemical reaction into physical movement of DNA. We further observed the time course of ATP hydrolysis by gp2 by determining structures of gp2:ATP complexes captured at various incubation time. These structures have made it possible to delineate, at atomic detail, the complete cycle of ATP hydrolysis of this viral DNA-packaging molecular motor.

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