scholarly journals Catalytic cycling of human mitochondrial Lon protease

2021 ◽  
Author(s):  
Inayathulla Mohammed ◽  
Kai A. Schmitz ◽  
Niko Schenck ◽  
Annika Topitsch ◽  
Timm Maier ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Thermal Unfolding ◽  
Lon Protease ◽  
Mitochondrial Matrix ◽  
Terminal Part ◽  
Brownian Ratchet ◽  
Conformational States ◽  
Threefold Symmetry ◽  
Substrate Protein

The mitochondrial Lon protease homolog (LonP1) hexamer controls mitochondrial health by digesting proteins from the mitochondrial matrix that are damaged or must be removed. Understanding how it is regulated requires characterizing its mechanism. Here, we show how human LonP1 functions, based on eight different conformational states that we determined by cryo-EM with a resolution locally extending to 3.6 Å for the best ordered states. LonP1 has a poorly ordered N-terminal part with apparent threefold symmetry, which apparently binds substrate protein and feeds it into its AAA+ unfoldase core. This translocates the extended substrate protein into a proteolytic cavity, in which we report an additional, previously unidentified Thr-type proteolytic center. Threefold rocking movements of the flexible N-terminal assembly likely assist thermal unfolding of the substrate protein. Our data suggest LonP1 may function as a sixfold cyclical Brownian ratchet controlled by ATP hydrolysis.

scholarly journals Mitochondrial Lon protease is a gatekeeper for proteins newly imported into the matrix

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yuichi Matsushima ◽  
Kazuya Takahashi ◽  
Song Yue ◽  
Yuki Fujiyoshi ◽  
Hideaki Yoshioka ◽  
...  
Keyword(s):  
Protease Activity ◽  
Matrix Protein ◽  
Protein Import ◽  
Atp Hydrolysis ◽  
Lon Protease ◽  
Mitochondrial Matrix ◽  
The Matrix ◽  
Specific Proteins ◽  
Aggregation Activity ◽  
Hydrolysis Activity

AbstractHuman ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

scholarly journals Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

2019 ◽  
Vol 116 (15) ◽  
pp. 7333-7342 ◽  
Author(s):  
Xiang Ye ◽  
Jiabei Lin ◽  
Leland Mayne ◽  
James Shorter ◽  
S. Walter Englander
Keyword(s):  
Hydrogen Exchange ◽  
Atp Hydrolysis ◽  
Physiological Solution ◽  
Mass Spectrometry Analysis ◽  
Structural Element ◽  
Spectrometry Analysis ◽  
Exchange Mass Spectrometry ◽  
Substrate Protein ◽  
Central Pore ◽  
The One

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

scholarly journals Lon protease: a novel mitochondrial matrix protein in the interconnection between drug-induced mitochondrial dysfunction and endoplasmic reticulum stress

2017 ◽  
Vol 174 (23) ◽  
pp. 4409-4429 ◽  
Author(s):  
Miriam Polo ◽  
Fernando Alegre ◽  
Angela B Moragrega ◽  
Lara Gibellini ◽  
Alberto Marti-Rodrigo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Mitochondrial Dysfunction ◽  
Matrix Protein ◽  
Lon Protease ◽  
Mitochondrial Matrix ◽  
Drug Induced

scholarly journals Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

2018 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Atp Binding ◽  
Conformational States ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Hexameric Atpase ◽  
Central Pore

AbstractThe 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals Setting the chaperonin timer: The effects of K+ and substrate protein on ATP hydrolysis

2008 ◽  
Vol 105 (45) ◽  
pp. 17334-17338 ◽  
Author(s):  
J. P. Grason ◽  
J. S. Gresham ◽  
L. Widjaja ◽  
S. C. Wehri ◽  
G. H. Lorimer
Keyword(s):  
Atp Hydrolysis ◽  
Substrate Protein

Utilization of positional isotope exchange experiments to evaluate reversibility of ATP hydrolysis catalyzed by Escherichia coli Lon proteaseThis paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Jennifer Thomas ◽  
Jennifer Fishovitz ◽  
Irene Lee
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Isotope Exchange ◽  
Atp Hydrolysis ◽  
Peer Review Process ◽  
Lon Protease ◽  
Peptidase Activity ◽  
E Coli ◽  
Atpase Reaction ◽  
Half Site

Lon protease, also known as protease La, is an ATP-dependent serine protease. Despite the presence of a proteolytic Ser–Lys dyad, the enzyme only catalyzes protein degradation in the presence of ATP. Lon possesses an intrinsic ATPase activity that is stimulated by protein and certain peptide substrates. Through sequence alignment and analysis, it is concluded that Lon belongs to the AAA+ protein family. Previous kinetic characterization of the ATPase domain of Escherichia coli Lon protease implicates a half-site reactivity model in which only 50% of the ATP bound to Lon are hydrolyzed to yield ADP; the remaining ATPase sites remain bound with ATP and are considered non-catalytic. In this model, it is implied that ATP hydrolysis is irreversible. To further evaluate the proposed half-site reactivity model, the reversibility of the ATPase activity of E. coli Lon was evaluated by positional isotope exchange experiments. The ATPase reactions were conducted in the 18O-enriched buffer such that the extent of 18O incorporation into inorganic phosphate generated from ATP hydrolysis could be used to evaluate the extent of reversibility in ATP hydrolysis. Collectively, our experimental data reveal that the ATPase reaction catalyzed by E. coli Lon in the presence and absence of peptide substrate that stimulated the enzyme’s ATPase activity is irreversible. Therefore, the half-site ATPase reactivity of E. coli Lon is validated, and can be used to account for the kinetic mechanism of the ATP-dependent peptidase activity of the enzyme.

Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis

Biochemistry ◽  
1987 ◽  
Vol 26 (1) ◽  
pp. 314-323 ◽  
Author(s):  
Vincent A. Barnett ◽  
David D. Thomas
Keyword(s):  
Steady State ◽  
Atp Hydrolysis ◽  
Conformational States

scholarly journals Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Katsuaki Oyama ◽  
Chihiro Azai ◽  
Kaori Nakamura ◽  
Syun Tanaka ◽  
Kazuki Terauchi
Keyword(s):  
Atp Hydrolysis ◽  
Circadian Oscillation ◽  
Conformational States

scholarly journals ROLE OF EDTA AND METALS IN MITOCHONDRIAL CONTRACTION

1964 ◽  
Vol 23 (1) ◽  
pp. 9-19 ◽  
Author(s):  
William S. Lynn ◽  
Sydney Fortney ◽  
Rose H. Brown
Keyword(s):  
Atp Hydrolysis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Mitochondrial Membranes ◽  
Semipermeable Membranes ◽  
Microscopic Appearance ◽  
Hydration And Dehydration ◽  
Mitochondrial Contraction

Studies comparing the state of hydration and dehydration of rat liver mitochondria to their content of ATP, Ca, and fatty acid, along with the rate of ATP hydrolysis, as well as microscopic appearance of mitochondria, have led to the following generalizations: 1. The competition between cationic translocations and water translocation for the available chemical energy (ATP) determines under many circumstances the water content of mitochondria. 2. Swelling of mitochondria by electron transport substrates is an example of the activation of the cationic translocations at the expense of water translocation. 3. Electron micrographic studies are interpreted to indicate that EDTA alone can cause condensation and dehydration of the mitochondrial matrix. However, both EDTA and substrate are necessary to remove appreciable quantities of water from mitochondrial intramembranous spaces. 4. Since the data in the accompanying report indicated that EDTA, in the absence of energy, decreased the permeability of mitochondrial membranes, it appears likely that ballooning of intramembranous spaces, following addition of EDTA, represents trapping of water between two semipermeable membranes following dehydration of mitochondrial matrix.

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