Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

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

eLife ◽

10.7554/elife.52774 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 24

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.

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Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells

Biochemical Journal ◽

10.1042/bj3640041 ◽

2002 ◽

Vol 364 (1) ◽

pp. 41-47 ◽

Cited By ~ 145

Author(s):

Maria RUZZENE ◽

Daniele PENZO ◽

Lorenzo A. PINNA

Keyword(s):

Protein Kinase ◽

Protein Kinase Ck2 ◽

Specific Inhibitor ◽

Specific Protein ◽

Jurkat Cells ◽

Similar Data ◽

Caspase Cleavage ◽

Protein Substrates ◽

Kinase Ck2

Incubation of Jurkat cells with 4,5,6,7-tetrabromobenzotriazole (TBB), a specific inhibitor of protein kinase CK2, induces dose-and time-dependent apoptosis as judged by several criteria. TBB-promoted apoptosis is preceded by inhibition of Ser/Thr phosphorylation of haematopoietic lineage cell-specific protein 1 (HS1) and is accompanied by caspase-dependent fragmentation of the same protein. Both effects are also observable if apoptosis is promoted by anti-Fas antibodies and by etoposide. Moreover, in vitro experiments show that HS1, once phosphorylated by CK2, becomes refractory to cleavage by caspase-3. These findings, in conjunction with similar data in the literature concerning two other CK2 protein substrates, Bid and Max, suggest that CK2 may play a general anti-apoptotic role through the generation of phosphorylated sites conferring resistance to caspase cleavage.

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Toward an understanding of the Cdc48/p97 ATPase

F1000Research ◽

10.12688/f1000research.11683.1 ◽

2017 ◽

Vol 6 ◽

pp. 1318 ◽

Cited By ~ 50

Author(s):

Nicholas Bodnar ◽

Tom Rapoport

Keyword(s):

Atp Hydrolysis ◽

Critical Role ◽

Ring Structure ◽

Cellular Systems ◽

Aaa Atpase ◽

Double Ring ◽

Terminal Domain ◽

D2 Domain ◽

Central Pore

A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER)-associated protein degradation (ERAD), Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2). Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN) have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB). Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF). Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

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Cataract-associated deamidations on the surface of γS-crystallin increase protein unfolding and flexibility at distant regions

10.1101/646083 ◽

2019 ◽

Author(s):

Heather M. Forsythe ◽

Calvin Vetter ◽

Kayla Ann Jara ◽

Patrick N. Reardon ◽

Larry L. David ◽

...

Keyword(s):

Hydrogen Exchange ◽

Protein Unfolding ◽

Structural Changes ◽

Human Lens ◽

Relaxation Dynamics ◽

Amide Hydrogen Exchange ◽

Terminal Domain ◽

Age Related ◽

Surface Loops

AbstractDeamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from cataractous lenses. However, the mechanism by which deamidation causes proteins to become insoluble is not known, because of only subtle structural changes observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues. We used heteronuclear NMR spectroscopy to measure their amide hydrogen exchange and 15N relaxation dynamics to identify regions with significantly increased dynamics compared to wildtype-γS. Changes in dynamics were localized to the C-terminal domain, particularly to helix and surface loops distant from the mutation sites. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility.

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

10.1101/704999 ◽

2019 ◽

Cited By ~ 4

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

SUMMARYClpXP 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.

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In Vitro and In Vivo Activities of Macrolide Derivatives against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1447-1454.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1447-1454 ◽

Cited By ~ 136

Author(s):

Kanakeshwari Falzari ◽

Zhaohai Zhu ◽

Dahua Pan ◽

Huiwen Liu ◽

Poonpilas Hongmanee ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Vero Cells ◽

Infection Model ◽

Gram Positive Bacteria ◽

Fold Reduction ◽

Dependent Inhibition ◽

Low Cytotoxicity ◽

Aerosol Infection

ABSTRACTExisting macrolides have never shown definitive clinical efficacy in tuberculosis. Recent reports suggest that ribosome methylation is involved in macrolide resistance inMycobacterium tuberculosis, a mechanism that newer macrolides have been designed to overcome in gram-positive bacteria. Therefore, selected macrolides and ketolides (descladinose) with substitutions at positions 9, 11,12, and 6 were assessed for activity againstM. tuberculosis, and those with MICs of ≤4 μM were evaluated for cytotoxicity to Vero cells and J774A.1 macrophages. Several compounds with 9-oxime substitutions or aryl substitutions at position 6 or on 11,12 carbamates or carbazates demonstrated submicromolar MICs. For the three macrolide-ketolide pairs, macrolides demonstrated superior activity. Four compounds with low MICs and low cytotoxicity also effected significant reductions in CFU in infected macrophages. Active compounds were assessed for tolerance and the ability to reduce CFU in the lungs of BALB/c mice in an aerosol infection model. A substituted 11,12 carbazate macrolide demonstrated significant dose-dependent inhibition ofM. tuberculosisgrowth in mice, with a 10- to 20-fold reduction of CFU in lung tissue. Structure-activity relationships, some of which are unique toM. tuberculosis, suggest several synthetic directions for further improvement of antituberculosis activity. This class appears promising for yielding a clinically useful agent for tuberculosis.

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Lipid-specific protein oligomerization is regulated by two interfaces in Marburg virus matrix protein VP40

10.1101/2020.11.13.381350 ◽

2020 ◽

Author(s):

Souad Amiar ◽

Monica L. Husby ◽

Kaveesha J. Wijesinghe ◽

Stephanie Angel ◽

Nisha Bhattarai ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Mechanistic Model ◽

Ultrastructural Localization ◽

Specific Protein ◽

Marburg Virus ◽

Protein Oligomerization ◽

Matrix Assembly ◽

Terminal Domain

SummaryMarburg virus major matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane and undergo a dynamic and extensive self-oligomerization into the structural matrix layer which confers the virion shape and stability. Using a myriad of in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrated the importance of each interface in the mVP40 matrix assembly through protein trafficking to the plasma membrane and homo-multimerization that induced protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. A novel APEX-TEM method was employed to closely assess the ultrastructural localization of and formation of viral particles for wild type and mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly.

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Multistep substrate binding and engagement by the AAA+ ClpXP protease

10.1101/2020.05.04.076331 ◽

2020 ◽

Cited By ~ 1

Author(s):

Reuben A. Saunders ◽

Benjamin M. Stinson ◽

Tania A. Baker ◽

Robert T. Sauer

Keyword(s):

Atp Hydrolysis ◽

Substrate Binding ◽

Specific Protein ◽

Dissociation Kinetics ◽

E Coli ◽

Protein Substrates ◽

Channel Opening ◽

Initial Substrate ◽

Domains Of Life ◽

Initial Binding

AbstractE. coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.SignificanceAAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show that E. coli ClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.

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Structural and Functional Characterization of Rv2966c Protein Reveals an RsmD-like Methyltransferase from Mycobacterium tuberculosis and the Role of Its N-terminal Domain in Target Recognition

Journal of Biological Chemistry ◽

10.1074/jbc.m110.200428 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19652-19661 ◽

Cited By ~ 22

Author(s):

Atul Kumar ◽

Kashyap Saigal ◽

Ketan Malhotra ◽

Krishna Murari Sinha ◽

Bhupesh Taneja

Keyword(s):

Mycobacterium Tuberculosis ◽

Target Recognition ◽

Functional Characterization ◽

Cellular Growth ◽

Functional Domain ◽

E Coli ◽

Terminal Domain ◽

16 S Rrna

Nine of ten methylated nucleotides of Escherichia coli 16 S rRNA are conserved in Mycobacterium tuberculosis. All the 10 different methyltransferases are known in E. coli, whereas only TlyA and GidB have been identified in mycobacteria. Here we have identified Rv2966c of M. tuberculosis as an ortholog of RsmD protein of E. coli. We have shown that rv2966c can complement rsmD-deleted E. coli cells. Recombinant Rv2966c can use 30 S ribosomes purified from rsmD-deleted E. coli as substrate and methylate G966 of 16 S rRNA in vitro. Structure determination of the protein shows the protein to be a two-domain structure with a short hairpin domain at the N terminus and a C-terminal domain with the S-adenosylmethionine-MT-fold. We show that the N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. Deletion of the N-terminal domain prevents binding to nucleic acid substrates, and the truncated protein fails to carry out the m2G966 methylation on 16 S rRNA. The N-terminal domain also binds DNA efficiently, a property that may be utilized under specific conditions of cellular growth.

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Multistep substrate binding and engagement by the AAA+ ClpXP protease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010804117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28005-28013 ◽

Cited By ~ 1

Author(s):

Reuben A. Saunders ◽

Benjamin M. Stinson ◽

Tania A. Baker ◽

Robert T. Sauer

Keyword(s):

Kinetic Data ◽

Atp Hydrolysis ◽

Substrate Binding ◽

Specific Protein ◽

Stopped Flow ◽

Dissociation Kinetics ◽

Protein Substrates ◽

Channel Opening ◽

Initial Substrate ◽

Quenching Assay

Escherichia coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

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