scholarly journals Structural basis for distinct operational modes and protease activation in AAA+ protease Lon

2020 ◽  
Vol 6 (21) ◽  
pp. eaba8404 ◽  
Author(s):  
Mia Shin ◽  
Cristina Puchades ◽  
Ananya Asmita ◽  
Neha Puri ◽  
Eric Adjei ◽  
...  
Keyword(s):  
Active Sites ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Protein Substrates ◽  
Left Handed ◽  
Protease Activation ◽  
Active Protease ◽  
Mechanistic Basis ◽  
Aaa Protein ◽  
Operational Modes

Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo–electron microscopy to determine structures of the Yersinia pestis Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, “open” spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed “closed” conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.

scholarly journals Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

2020 ◽  
Author(s):  
Mia Shin ◽  
Edmond R. Watson ◽  
Scott J. Novick ◽  
Patrick Griffin ◽  
R. Luke Wiseman ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Basis ◽  
Proteolytic Activation ◽  
Mitochondrial Regulation ◽  
Cryo Electron Microscopy ◽  
Transport Chain ◽  
Protease Activation ◽  
Health And Disease ◽  
Aaa Protein ◽  
Spiral Staircase

AbstractThe human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

scholarly journals Structural basis of the XPB–Bax1 complex as a dynamic helicase–nuclease machinery for DNA repair

2020 ◽  
Vol 48 (11) ◽  
pp. 6326-6339 ◽  
Author(s):  
Kevin DuPrez ◽  
Feng He ◽  
Zhenhang Chen ◽  
Eduardo Hilario ◽  
Li Fan
Keyword(s):  
Dna Repair ◽  
Protein Interactions ◽  
Active Sites ◽  
Atp Hydrolysis ◽  
Excision Repair ◽  
Dna Lesions ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Nucleotide Excision ◽  
First Time

Abstract Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

scholarly journals Structures of the human LONP1 protease reveal regulatory steps involved in protease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mia Shin ◽  
Edmond R. Watson ◽  
Albert S. Song ◽  
Jeffrey T. Mindrebo ◽  
Scott J. Novick ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Cryo Electron Microscopy ◽  
Transport Chain ◽  
Protease Activation ◽  
Active Protease ◽  
Health And Disease ◽  
Aaa Protein ◽  
Spiral Staircase

AbstractThe human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

scholarly journals Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

2021 ◽  
Vol 7 (2) ◽  
pp. eabd4413
Author(s):  
Jung-Hoon Lee ◽  
Daniel Bollschweiler ◽  
Tillman Schäfer ◽  
Robert Huber
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Active Sites ◽  
Chromatin Modification ◽  
Structural Basis ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts ◽  
Lysine Residues ◽  
Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

scholarly journals Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bertrand Beckert ◽  
Elodie C. Leroy ◽  
Shanmugapriya Sothiselvam ◽  
Lars V. Bock ◽  
Maxim S. Svetlov ◽  
...  
Keyword(s):  
Antibiotic Resistance Genes ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

scholarly journals FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Proteolytic Activity ◽  
Guinea Pigs ◽  
Protease Activity ◽  
Amino Acid Esters ◽  
Protein Substrates ◽  
Protease Activation ◽  
Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

scholarly journals Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins

2019 ◽  
Author(s):  
Liang Qin ◽  
Fredj Ben Bdira ◽  
Yann G. J. Sterckx ◽  
Alexander N. Volkov ◽  
Jocelyne Vreede ◽  
...  
Keyword(s):  
Dna Binding ◽  
Electrostatic Interactions ◽  
Intramolecular Interactions ◽  
Structural Basis ◽  
Binding Properties ◽  
Family Protein ◽  
Low Salt ◽  
Functional States ◽  
Mechanistic Basis ◽  
Dna Binding Properties

AbstractH-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.Here, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

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 Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Simon Jenni ◽  
Benjamin M Stinson ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

scholarly journals Structural Basis of Binding and Rationale for the Potent Urease Inhibitory Activity of Biscoumarins

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Muhammad Arif Lodhi ◽  
Sulaiman Shams ◽  
Muhammad Iqbal Choudhary ◽  
Atif Lodhi ◽  
Zaheer Ul-Haq ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Active Sites ◽  
Docking Simulation ◽  
Cysteine Residue ◽  
Structural Basis ◽  
Bacillus Pasteurii ◽  
Nickel Ions ◽  
Jack Bean ◽  
Uv Visible

Urease belongs to a family of highly conserved urea-hydrolyzing enzymes. A common feature of these enzymes is the presence of two Lewis acid nickel ions and reactive cysteine residue in the active sites. In the current study we examined a series of biscoumarins1–10for their mechanisms of inhibition with the nickel containing active sites of Jack bean andBacillus pasteuriiureases. All these compounds competitively inhibited Jack bean urease through interaction with the nickel metallocentre, as deduced from Michaelis-Menten kinetics, UV-visible absorbance spectroscopic, and molecular docking simulation studies. Some of the compounds behaved differently in case ofBacillus pasteuriiurease. We conducted the enzyme kinetics, UV-visible spectroscopy, and molecular docking results in terms of the known protein structure of the enzyme. We also evaluated possible molecular interpretations for the site of biscoumarins binding and found that phenyl ring is the major active pharmacophore. The excellent in vitro potency and selectivity profile of the several compounds described combined with their nontoxicity against the human cells and plants suggest that these compounds may represent a viable lead series for the treatment of urease associated problems.

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