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[11] Coupled assay of Na+,K+-ATPase activity
... To assess ATP hydrolysis at normal or elevated temperatures, we measured the ATPase activities of HSP90C and SecA1. Utilizing NADH-coupled spectroscopic analysis [43], we investigated their basal ATP turnover rates at a saturated 5 mM ATP, approximating maximum ATP hydrolysis turnover rates. Notably, the turnover rates for HSP90C and SecA1 were determined to be 0.880 ± 0.074 min −1 and 2.046 ± 0.124 min −1 , respectively ( Figure 4). ...
... ATP-hydrolysis activity was measured using the coupled-NADH method as mentioned previously [43]. Purified HSP90C, SecA1, or PsbO1 proteins were mixed in equimolar compositions (1.41 µM) in the assay reaction mixture consisting of 25 HEPES (pH 7.5), 5 mM MgCl 2 , 500 mM KCl, 0.03% Tween 20, 10% glycerol, 200 µM NADH, 3 mM phosphoenol pyruvate, 15.7U of pyruvate kinase and 24.5U of lactate dehydrogenase (MilliporeSigma, Oakville, Canada). ...
The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis within chloroplasts and participates in protein translocation processes. While existing studies have revealed HSP90C’s direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unraveled potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client pre-protein, we studied the intricacies of HSP90C’s possible involvement in pre-protein translocation via the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction, possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C’s engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane.
... LmRUVBLs ATPase activity was assessed using the ATP/NADH coupled assay [25]. The reactions were carried out in 96-well plates in a buffer containing 25 mM Tris-HCl, 500 mM NaCl, 8 mM MgCl 2 , pH 8.0 mixed with 3 mM phosphoenolpyruvate, 0.3 mM NADH, 40 U/mL pyruvate kinase and 58 U/mL lactate dehydrogenase. ...
... Indeed, the solubilization of RUVBLs from parasites is challenging. For instance, both RUVBL1 [25] and RUVBL3, an analog of RUVBL2, [27] from P. falciparum required the assistance of chaperones to increase their solubility. ...
ATPases belonging to the AAA + family are associated with diverse cellular activities and are mainly characterized by the nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA + proteins have a range of functions, from DNA replication to protein degradation. Rvbs are AAA + ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.
... ATPase activity measurement. The effect of TF on ClpX ATPase activity was measured using a coupled assay 69 ClpP was added at 1.2 µM, and λO at 3.9 µM if needed. After preincubation for 3 min, 5 mM ATP was added to start the reaction and the change in absorbance at 340 nm was measured for 20 min at 37°C. ...
... The extinction coefficient used for NADH is 6220 M −1 cm −1 (ref. 69 ). Each reaction was performed at least three times. ...
A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig , the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone–protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
... To assess if arginine finger variants of Rvb1 and Rvb2 impact the enzyme's activity, we characterized the wild-type and variant Rvb1/2 complexes in vitro. We first measured the steady-state ATPase activity of Rvb1/2 and its R/A variants using an NADHcoupled ATPase assay under multiple turnover conditions (56). Using the indicated ATP concentrations in these assays, we obtained k cat and K M values ( Figure S4A). ...
The evolutionarily conserved AAA ⁺ ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA ⁺ subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA ⁺ subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex.
Significance
AAA ATPases constitute a large family of proteins involved in various essential cellular functions in living organisms in all kingdoms of life. Members of this family typically form homo or hetero multimers that convert the energy from ATP hydrolysis to mechanical work. How the conserved features of AAA ATPases relay the energy from ATP hydrolysis to other functional domains of the complex remains largely unknown. Here, using arginine finger variants of Rvb1 and Rvb2, two evolutionarily conserved closely related AAA ⁺ ATPases that form a heterohexameric complex, we reveal how individual protomers in a heteromeric complex can uniquely contribute to the overall function of the complex and how changes in the ATP binding site can be relayed to distal functional domains.
... The ATPase activity assay was performed as described before 33 using an NADH-coupled assay 76 . The assay was performed in a 96-well plate and carried out in a buffer containing 50 mM Tris (pH 7.5), 200 mM NaCl and 5 mM MgCl2 in the presence of 1-2 mM NADH, 2.5 mM ATP, 2.5 mM phosphoenolpyruvate, 50 μg/ml pyruvate kinase, and 50 μg/ml lactate dehydrogenase. ...
New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have created a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode-of-action of the degraders on a challenging essential target GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.
... ATP hydrolysis was measured using a coupled enzymatic reaction (Norby 1988) in which NADH oxidation to NAD+ reduces absorbance at 340 nm (∆ε = 6.22 mM -¹ cm -¹) using a SpectraMax M5 plate reader and a 384-well assay plate (Corning,3575) ...
AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.
... ATP hydrolytic activity was measured using an NADH-coupled assay 42 adapted for plate readers 43 , as previously described 19 . Each 50 μL reaction is made up of 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 0.05% DDM, 200mM NADH (Merck, 10128023001), 20 U/mL lactic dehydrogenase (Merck, L1254), 100 U/mL pyruvate kinase (Merck, P9136), 0.5 mM phosphoenolpyruvate (Merck, P7127) and various ATP (Merck, A7699) concentrations. ...
Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter associated with distinct heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers a molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies.
... The ATPase activity of the samples was measured as described 43 The ATPase-regeneration system (0.3 mM NADH, 3 mM PEP, 20 ng/mL PK, 0.017 mg/mL lactate dehydrogenase (LDH) and 2 mM ATP) was added to initiate the reaction. ATP consumption rates (μmol ATP/min) were calculated from slopes of the A 340 decay curves that presented a linear absorbance decline using the extinction coefficient of NADH (E 340 6220/M/cm). ...
J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform. Cryoelectron microscopy and mutational studies reveal that different domains are involved in self-association. Oligomer dissociation into dimers potentiates its interaction with unfolded client proteins. The J-domains are accessible to Hsc70 within the tubular structure. They allow binding of closely spaced Hsc70 molecules that could be transferred to the unfolded substrate for its cooperative remodelling, explaining the efficient recovery of DNAJA2-bound clients. The disordered C-terminal domain, comprising the last 52 residues, regulates its holding activity and productive interaction with Hsc70. These in vitro findings suggest that the association equilibrium of DNAJA2 could regulate its interaction with client proteins and Hsc70.
... Degradation of GFP-substrate fusions was monitored by loss of 511-nm emission following excitation at 450 nm. ATPase assays utilized 1 mM ClpC1, 10 mM ATP, and an NADH-coupled ATP regeneration system (89). Consumption of ATP was followed by monitoring the decrease in NADH absorbance at 340 nm. ...
The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coim-munoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfol-dase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.
... Coupled enzyme ATPase assays. ATPase activity was measured using a coupled enzyme ATPase assay (88). Purified His-tagged ConE, wild-type or mutant (varied from 0.2 to 10 mM, in monomer equivalent), was analyzed at room temperature in dialysis buffer N with 2 or 10 mM magnesium chloride or in dialysis buffer K with 10 mM magnesium acetate. ...
Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICEBs1, an integrative and conjugative element in Bacillus subtilis. ConE, encoded by ICEBs1, is a member of the VirB4 family of ATPases, the most conserved component of T4SSs. ConE is required for conjugation and localizes to the cell membrane, predominantly at the cell poles. In addition to Walker A and B boxes, VirB4 homologs have conserved ATPase motifs C, D, and E. Here, we created alanine substitutions in five conserved residues within or near ATPase motifs in ConE. Mutations in all five residues drastically decreased conjugation frequency but did not affect ConE protein levels or localization, indicating that an intact ATPase domain is critical for DNA transfer. Purified ConE is largely monomeric with some oligomers and lacks enzymatic activity, suggesting that ATP hydrolysis may be regulated or require special solution conditions. Finally, we investigated which ICEBs1 T4SS components interact with ConE using a bacterial two-hybrid assay. ConE interacts with itself, ConB, and ConQ, but these interactions are not required to stabilize ConE protein levels and largely do not depend on conserved residues within the ATPase motifs of ConE. The structure-function characterization of ConE provides more insight into this conserved component shared by all T4SSs. IMPORTANCE Conjugation is a major form of horizontal gene transfer and involves the transfer of DNA from one bacterium to another through the conjugation machinery. Conjugation contributes to bacterial evolution by disseminating genes involved in antibiotic resistance, metabolism, and virulence. Here, we characterized ConE, a protein component of the conjugation machinery of the conjugative element ICEBs1 of the bacterium Bacillus subtilis. We found that mutations in the conserved ATPase motifs of ConE disrupt mating but do not alter ConE localization, self-interaction, or levels. We also explored which conjugation proteins ConE interacts with and whether these interactions contribute to stabilizing ConE. Our work contributes to the understanding of the conjugative machinery of Gram-positive bacteria.
... ATPase activity assay. The ATP-hydrolysis rate of TRiC was measured by performing an NADH-coupled assay 72 . In general, in this assay, each ATP-hydrolysis event allows for a pyruvate kinase (PK)-catalyzed conversion of one molecule of phosphoenolpyruvate into pyruvate, with pyruvate then converted to lactate by Llactate dehydrogenase, resulting in oxidation of a single NADH molecule. ...
The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin—with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.
... ATPase activity assay ATP hydrolysis rates of TRiC with plp2 and β-actin were measured by performing an NADH-coupled assay (71). In this assay, each ATP hydrolysis event allows conversion of one molecule of phosphoenolpyruvate into pyruvate by pyruvate kinase. ...
The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers. Notably, we captured an unprecedented TRiC-plp2-tubulin complex in the closed S3 state, engaged with a folded full-length β-tubulin and loaded with a guanosine triphosphate, and a plp2 occupying opposite rings. Another closed S4 state revealed an actin in the intermediate folding state and a plp2. Accompanying TRiC ring closure, plp2 translocation could coordinate substrate translocation on the CCT6 hemisphere, facilitating substrate stabilization and folding. Our findings reveal the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the biogenesis machinery TRiC and plp2 and extend our understanding of the links between cytoskeletal proteostasis and related human diseases.
... ATPase activity was measured using the pyruvate kinasecoupled assay [59]. The reaction mixture contained 50 mM TRIS/HCl, pH 8.0, 150 mM NaCl, 15 mM MgCl 2 , 2.5 mM ATP, 1 mM phosphoenolpyruvate, 0.3 mM NADH, 12 U/mL pyruvate kinase, and 12 U/mL lactate dehydrogenase. ...
Background
NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.
Results
We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.
Conclusions
Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.
... ATP activity of ClpB mutants was measured using a coupled colorimetic assay 54 ...
AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate-proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using smFRET, we study the allosteric coupling between the pore loops and the two nucleotide binding domains of ClpB (NBD1-2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis, but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines.
Statement of Significance
Molecular machines function by coupling the energy of ATP hydrolysis to mechanical motion. How this coupling occurs and what timescales are involved remains an open question. In this study, we use a powerful single-molecule FRET technique to measure the real-time dynamics of pore loops, which are essential protein-translocating elements of the ATP-dependent disaggregation machine ClpB. Using a series of mutations of the ATP-hydrolysis motifs of ClpB, we find that, although the motions of these pore loops take place on the microsecond time scale, they are markedly affected by the much slower changes in the nucleotide state of the machine. Generally, this study shows that protein machines, such as ClpB, are wired to harness ATP binding and hydrolysis to allosterically affect distal events, such as the function-related mechanics of pore-loops.
... The ATP hydrolysis rate of reconstituted proteasomes was measured using a NADH-coupled kinetic assay (Nørby, 1988). Proteasomes were reconstituted under base-limiting conditions (400 nM CP, 200 nM base, 800 nM Rpn10, 800 nM WT or sem1Δ (which was not certified by peer review) is the author/funder. ...
The 26S proteasome is an ∼70 subunit ATP-dependent chambered protease that destroys proteins via multiple highly coordinated processing steps. The smallest and only intrinsically disordered proteasome subunit, Sem1 (DSS1 in metazoans), is critical for efficient substrate degradation despite lacking obvious enzymatic activities and being located far away from the proteasome’s catalytic centers. Dissecting its role in proteolysis using cell-based approaches has been challenging because Sem1 also controls proteasome function indirectly via its role in proteasome biogenesis. To circumvent this challenge, we reconstituted Sem1-deficient proteasomes in vitro from purified components and systematically dissected its impact on distinct processing steps. Whereas most substrate processing steps are independent of Sem1, ATP-dependent unfolding is stimulated several-fold. Using structure-guided mutagenesis and engineered protein crosslinking, we demonstrate that Sem1 allosterically regulates ATP-dependent substrate unfolding via a distal conformation-dependent intersubunit contact. Together, this work reveals how a small, unstructured subunit comprising < 0.4% the total size of the proteasome can augment substrate processing from afar, and reveals a new allosteric pathway in controlling proteolysis.
... Samples were quenched at indicated times with SDS loading buffer, separated by SDS-PAGE, and visualized by Coomassie staining. ATP-hydrolysis rates were measured using a continuous spectrophotometric assay (Norby, 1988). ...
Targeted protein degradation plays important roles in stress responses in all cells. In E. coli, the membrane‐bound AAA+ FtsH protease degrades cytoplasmic and membrane proteins. Here, we demonstrate that FtsH degrades cyclopropane fatty acid (CFA) synthase, whose synthesis is induced upon nutrient deprivation and entry into stationary phase. We find that neither the disordered N‐terminal residues nor the structured C‐terminal residues of the kinetically stable CFA‐synthase dimer are required for FtsH recognition and degradation. Experiments with fusion proteins support a model in which an internal degron mediates FtsH recognition as a prelude to unfolding and proteolysis. These findings elucidate the terminal step in the life cycle of CFA synthase and provide new insight into FtsH function. The cellular proteome is in a constant state of flux, as changes in the environment necessitate rapid functional responses, often via mechanisms involving protein degradation. FtsH, a membrane‐bound AAA+ protease, degrades cyclopropane fatty acid synthase, an enzyme that is transiently upregulated upon entrance into stationary phase. Interestingly, FtsH appears to utilize an internal sequence of CFA synthase for substrate recognition rather than degrons at either the N‐ or C‐terminus.
... ATPase assays. ATPase assays were conducted using an NADH-coupled assay 46 . Gyrase heterotetramer was formed as described above for the supercoil relaxation assays. ...
The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity. Evybactin is an antimicrobial natural product that targets DNA gyrase, where it binds to a site overlapping with synthetic thiophene poisons and exerts selectivity for Mycobacterium tuberculosis via its transport mechanism into the cell.
... ATP hydrolysis rates of TRiC with plp2 and β-actin were measured by performing an NADH coupled assay (69). In this assay, each ATP hydrolysis event allows conversion of one molecule of phosphoenolpyruvate into pyruvate by pyruvate kinase. ...
The eukaryotic chaperonin TRiC/CCT assists the folding of ~10% cytosolic proteins. The essential cytoskeletal proteins tubulin and actin are the obligate substrates of TRiC and their folding involves cochaperone and co-factors. Here, through cryo-EM analysis, we present a more complete picture of yeast TRiC-assisted tubulin and actin folding in the ATPase-cycle, under the coordination of cochaperone plp2. Our structures revealed that in the open C1 and C2 states, plp2 and substrates tubulin/actin engage with TRiC inside its chamber, one per ring. Noteworthy, we captured a ternary TRiC-plp2-tubulin complex in the closed C3 state, engaged with a full-length β-tubulin in the native folded state even loaded with a GTP, and with a plp2 occupying the opposite ring, not reported before. Another closed C4 state revealed an actin in the intermediate state of folding and a plp2 occupying the other ring. Intriguingly, along with TRiC ring closure, we captured a large translocation of plp2 within TRiC chamber coordinating substrate translocation on the CCT6 hemisphere, potentially facilitating substrate stabilization and folding. Our findings provide structural insights into the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the complex biogenesis machinery TRiC and plp2, and could extend our understanding on the links between cytoskeletal proteostasis and related human diseases.
... A 10Â pyruvate kinase-lactose dehydrogenase (PK-LDH) mixture was prepared in water that contained phosphoenolpyruvate (10108294001, 37.5 mM; PEP; Roche), NADH (10107735001, 7.5 mM; Roche), PK (P9135; Sigma), and LDH (10127230001, 100 U/mL each; Roche) (60). Final reaction mixtures contained 50 nM Gsk, 5 mM ATP, 1 mM ADG or guanosine in reaction buffer (40 mM HEPES-Na, pH 7.4, 150 mM KCl, 10 mM MgCl 2 , and 1 mM TCEP). ...
Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites.
... The integrity and purity of proteins were evaluated by SDS-PAGE. ATPase activity was measured as reported previously (Nørby, 1988). ...
Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 coreceptor, support a model in which the exportomer tail anchored to the peroxisome membrane represses Atg36 phosphorylation on Pex3 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.
... The ATPase activities of WT Hsp104 or Hsp104 variants (3 μM) were measured using a NADH-coupled colorimetric assay [98] by measuring the decrease of NADH absorption at 340 nm. The reactions were carried out in 50 mM of HEPES pH 7.4, 50 mM of KCl, 10 mM of MgCl 2 and 10 mM of DTT buffer supplemented with 1.8 mM of phosphoenolpyruvate, 0.5 mM of NADH, 50 μgÁmL −1 of pyruvate kinase, 50 μgÁmL −1 of lactate dehydrogenase and 3.5 mM of ATP [99]. ...
Heat shock protein 104 (Hsp104) protein disaggregases are powerful molecular machines that harness the energy derived from ATP binding and hydrolysis to disaggregate a wide range of protein aggregates and amyloids, as well as to assist in yeast prion propagation. Little is known, however, about how Hsp104 chaperones recognize such a diversity of substrates, or indeed the contribution of the substrate‐binding N‐terminal domain (NTD) to Hsp104 function. Herein, we present a NMR spectroscopy study, which structurally characterizes the Hsp104 NTD‐substrate interaction. We show that the NTD includes a substrate‐binding groove that specifically recognizes exposed hydrophobic stretches in unfolded, misfolded, amyloid and prion substrates of Hsp104. In addition, we find that the NTD itself has chaperoning activities which help to protect the exposed hydrophobic regions of its substrates from further misfolding and aggregation, thereby priming them for threading through the Hsp104 central channel. We further demonstrate that mutations to this substrate‐binding groove abolish Hsp104 activation by client proteins and keep the chaperone in a partially inhibited state. The Hsp104 variant with these mutations also exhibited significantly reduced disaggregation activity and cell survival at extreme temperatures. Together, our findings provide both a detailed characterization of the NTD‐substrate complex and insight into the functional regulatory role of the NTD in protein disaggregation and yeast thermotolerance.
... Pyruvate kinase (PK) and lactate dehydrogenase (LDH) enzymes couple the ATP consumption to the oxidation of NADH, which strongly absorbs to 340 nm. Thus, decrease in absorbance at 340 nm is directly correlated to ATP hydrolysis (44). The RUVBL1-RUVBL2 ATPase activity was measured at a final concentration of 3 M (being considered monomers) in the presence or absence of 3 M ZNHIT2 C. ATPase assays of ZNHIT2 C alone and without both RUVBL1-RUVBL2 and ZNHIT2 C were included as controls. ...
Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1–RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1–RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.
... NKA activity was estimated as ATP cleavage using the enzyme coupled assay method [83]. Incubation medium (2 mL) contained 130 mM NaCl, 20 mM KCl, 4 mM MgCl2, 3 mM ATP, 30 imidazole (pH 7.4), 1 mM phosphoenolpyruvate, 0.2 mM NADH, 0.18 мМ NADH and pyruvate kinase (600-1000 units/mL)/lactate dehydrogenase (900-1400 units/mL). ...
Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs’ steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin’s effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).
... In vitro ATP hydrolysis by ClpX was measured using an NADH-coupled assay as previously described [37]. A mixture of ClpX interacting protein (0-27.4 ...
The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.
... Coupled ATPase assays were performed based on the method described in Nørby (1988). 100 nM cohesin was incubated with 200 nM DN-NIPBL and 1 mM of a 75 bp ds DNA in ATPase reaction buffer (20 mM NaPO 4 pH pH 7.6, 50 mM NaCl, 20mM KCl, 2.5 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA, 2 mM ATP), supplemented with 0.795 mM NADH (Sigma), 5.5mM phosphoenol pyruvate (Sigma), 16 U/ml lactate dehydrogenase and 14 U/ml pyruvate kinase (Sigma). ...
Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin’s hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL “jumps ship” from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.
... The 595 integrity and purity of proteins were evaluated by SDS-PAGE. ATPase activity was measured as 596 reported previously (Nørby, 1988). 597 ...
Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 co-receptor, support a model in which exportomer represses Atg36 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.
... In vitro ATP hydrolysis by ClpX was measured using an NADH-coupled assay as previously described (Nørby, 1988). A mixture of ClpX interacting protein (0-27.4 ...
The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo . Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.
SUMMARY
This work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.
... where V 1 and V 2 are the maximum sliding rates for the two opposing remodeling directions. 514 ATPase assay 515 ATPase activity was measured using a coupled enzyme system in which regeneration 516 of hydrolyzed ATP is coupled to NADH oxidation as described previously (Nørby, 1988). 517 ...
The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.
... ATPase activity measurements were performed using the ATP/NADH coupled ATPase assay (Norby, 1988). The reactions consisted of 3 mM phosphoenolpyruvate, 0.2 mM NADH, 40 U/mL pyruvate kinase, 58 U/mL lactate dehydrogenase in the buffer K. Proteins of interest and ATP were added to a final volume of 150 mL and all the reaction components were incubated for 10 min at 37 C prior to ATP addition. ...
R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.
... Steady-state ATPase activity was measured using the assay described by Nørby [92]. Briefly, Assays were performed at 30 • C in 40 mM Hepes, pH 7.6, 50 mM KCl, 5 mM Mg acetate, and 1 mM ATP, using transparent 96-well plates with flat bottom (Sarstedt), a final volume of 200 µL and 6 µM Hsc70 or 2 µM Apg2. ...
Simple Summary
High levels of Heat shock proteins (Hsps) in specific cancers are usually linked to a poor prognosis, tumor progression, invasiveness, and resistance to treatment. Chaperone inhibition could therefore be toxic for cancer cells due to their high dependence on chaperone activity to survive. This study shows the potential to repurpose the small chemical compound pinaverium bromide, currently used to treat functional gastrointestinal disorders, as a possible antitumor drug since it displays a marked toxicity against two melanoma cell lines without affecting the viability of fibroblast and primary melanocytes. This compound interacts with structural regions shared by representatives of the Hsp70 and Hsp110 families, inhibiting the substrate remodeling ability of the Hsp70 system in vitro and in a cellular context.
Abstract
Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach.
... Steady-state ATPase assay. The ATPase rate of BiP and BiP mutants was determined by applying an ATP regenerating system in which the consumption of NADH directly corresponds to the hydrolysis of ATP 73 . The absorption of NADH was measured at 340 nm in a TECAN Spark microplate reader (Tecan) in Corn-ing® 384-well plates (#3640). ...
To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.
... The ATP activity of ClpB variants was measured using a coupled colorimetic assay. 61 ClpB or its mutants (1 μM total monomer concentration) were incubated with 2 mM ATP and an ATP regeneration system (2.5 mM phosphoenol pyruvate, 10 units/ml pyruvate kinase, 15 units/ml lactate dehydrogenase, 2 mM 1,4-dithioerythritol, 2 mM EDTA, 0.25 mM NADH) in 50 mM HEPES (pH 8), 50 mM KCl, and 0.01% Tween 20. For the experiments in the presence of the model substrate κcasein (Sigma-Aldrich), it was added to a final concentration of 50 μM. ...
ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.
... Degradation and ATPase assays were performed in 25 mM HEPES (pH 7.5), 5 mM KCl, 20 mM MgCl 2 , 10% glycerol, and 0.032% Igepal CA-630 as described (Baytshtok et al., 2016). ATPase rates at different temperatures were measured using a Spectramax M5 plate reader (Molecular Devices) by using an NADH-coupled assay (Nørby, 1988) with or without 50 μM I37A Arc-cp6 GFP-st11-ssrA. Using the Solver tool of Microsoft Excel, the temperature dependencies of ATPase rates were globally fitted to the equation max/(1+exp(ΔH/R·(1/ T M -1/T))), where max is the maximum ATPase rate, T is the temperature in Kelvin, T M is the temperature at 50% activity, ΔH is the enthalpy at T M , and R is the universal gas constant. ...
2020 The Author(s) At low temperatures, protein degradation by the AAA+ HslUV protease is very slow. New crystal structures reveal that residues in the intermediate domain of the HslU6 unfoldase can plug its axial channel, blocking productive substrate binding and subsequent unfolding, translocation, and degradation by the HslV12 peptidase. Biochemical experiments with wild-type and mutant enzymes support a model in which heat-induced melting of this autoinhibitory plug activates HslUV proteolysis.
... Peptidase assays used 2.85% dimethyl sulfoxide, 100 µM fluorogenic peptide (Bachem, Peptides International, or Enzo Life Sciences), and 0.4 μM ClpP tetradecamer (PaClpP 14 ), and cleavage was monitored by fluorescence with excitation/emission 380/460 nm at 30°C. Degradation of GFP-ssrA for Michaelis-Menten analysis was monitored as described using 0.54 μM PaClpP 14 , 0.1 μM ClpX 6 , and an NADH-coupled ATP regeneration system(Nørby, 1988; Oliveras et al., 2014). Enzymatic assays were monitored on a SpectraMax M5 microplate reader(Molecular Devices) in a black 384-well flat-bottom plate (Corning). ...
Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double‐ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen P. aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non‐canonical features in its N‐ and C‐terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late‐stage P. aeruginosa development.
... ATP hydrolysis by His-RUVBL1-RUVBL2 was measured in a continuous spectrophotometric pyruvate kinase-lactate dehydrogenase-coupled assay, based on the regeneration of the hydrolyzed ATP coupled to oxidation of NADH (Nørby, 1998). NADH absorbance at 340 nm was measured using a Jasco V-550 UV/VIS Spectrophotometer with a Jasco EHC-477T Temperature Controller and monitored using the ND-1000 and Spectra Manager software in time course experiments, and its decrease was used to determine ATP hydrolysis rates. ...
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.
Most of the fundamental processes of cells are mediated by proteins. However, the biologically- relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating mechanism, but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co- chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. Our analysis of all point mutations in Hsp90 identified 205 that dramatically slowed the growth of yeast harboring a second WT copy of Hsp90. 75% of the dominant negative mutations that we identified were located in a loop that closes over bound ATP. We analyzed a small panel of individual dominant mutations in this loop in detail. In this panel, addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. We examined the stability to proteolysis of glucocorticoid receptor (GR) as a model Hsp90 substrate. Upon expression of dominant negative Hsp90 variants, GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.
The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a “pincer-like” conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitinylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.
NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes the NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. We show that NorQ-catalyzed ATP hydrolysis, an intact VWA-domain in NorD and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that ″plugs″ the NorQ ring pore to the VWA domain of NorD with a protruding ″finger″ inserting through the pore, and suggest this binding mode to be general for MoxR/VWA couples. We present a tentative model for the mechanism of NorQD-catalyzed cNOR remodelling and suggest many of its features to be applicable to the whole MoxR/VWA family.
Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain with propensity to adopt α-helical structure interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.
The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated an essential role for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the nucleosomal acidic patch in the H2A.Z exchange reaction. Nucleosomes lacking acidic patch residues retain the ability to stimulate the ATPase activity of SWR1C, implicating a role in coupling the energy of ATP hydrolysis to H2A/H2B dimer eviction. A conserved arginine-rich region within the Swc5 subunit is identified that interacts with the acidic patch and is found to be essential for dimer exchange activity. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.
AAA (ATPases associated with diverse cellular activities) ATPases widely exist in many organisms and function in various organelles. However, there is little information about AAA ATPase functioning in endocytosis. In the filamentous fungus Aspergillus oryzae, we previously discovered a putative AAA ATPase AipA (AoAbp1 interacting protein) that would be involved in endocytosis. Here, we further examined the function of AipA and AoAbp1 in endocytosis using enhanced green fluorescent protein (EGFP)-tagged arginine permease AoCan1 as an endocytic marker. In the ΔaipA strain, endocytosis of AoCan1-EGFP was more facilitated than the control strain, suggesting that AipA negatively regulates endocytosis. Additionally, the localization of F-actin, visualized by Lifeact-EGFP, was concentrated at the hyphal tip in the ΔaipA strain than the control strain, suggesting that endocytosis was promoted due to enhanced actin polymerization in the ΔaipA strain. In contrast, in the ΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed compared with the control strain, suggesting that AoAbp1 positively functions in endocytosis. In addition, in the ΔaipAΔAoabp1 strain, endocytosis of AoCan1-EGFP was also delayed. AipA localized at the endocytic collar of the hyphal tip, only in the presence of AoAbp1, suggesting that AipA functions downstream of AoAbp1 in endocytosis. Moreover, we investigated the aipA-overexpressing strain, and found that endocytosis of AoCan1-EGFP was inhibited. Furthermore, we examined strains expressing aipAK542A or aipAE596Q, which decreased ATPase activity, in the backgrounds of complementation or overexpression, respectively, and found that AoCan1-EGFP endocytosis was promoted. These results suggested that AAA ATPase activity of AipA is important for its function in endocytosis.
In an event reminiscent of eukaryotic ubiquitination, the bacterial prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS) marks target proteins for proteasomal degradation by covalently attaching Pup, the bacterial tagging molecule. Yet, ubiquitin is released from its conjugated target following proteasome binding, whereas Pup enters the proteasome and remains conjugated to the target. Here, we report that although Pup can be degraded by the bacterial proteasome, it lacks favorable 20S core particle (CP) cleavage sites and is thus a very poor 20S CP substrate. Reconstituting the PPS in vitro, we demonstrate that during pupylated protein degradation, Pup can escape unharmed and remain conjugated to a target-derived degradation fragment. Removal of this degradation fragment by Dop, a depupylase, facilitates Pup recycling and re-conjugation to a new target. This study thus offers a mechanistic model for Pup recycling and demonstrates how a lack of protein susceptibility to proteasome-mediated cleavage can play a mechanistic role in a biological system.
To investigate monoallelic CLPB variants. Pathogenic variants in many genes cause congenital neutropenia. While most patients exhibit isolated hematological involvement, biallelic CLPB variants underlie a neurological phenotype ranging from nonprogressive intellectual disability to prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, 3-methylglutaconic aciduria, and neutropenia. CLPB was recently shown to be a mitochondrial refoldase; however, the exact function remains elusive.
We investigated six unrelated probands from four countries in three continents, with neutropenia and a phenotype dominated by epilepsy, developmental issues, and 3-methylglutaconic aciduria with next-generation sequencing.
In each individual, we identified one of four different de novo monoallelic missense variants in CLPB. We show that these variants disturb refoldase and to a lesser extent ATPase activity of CLPB in a dominant-negative manner. Complexome profiling in fibroblasts showed CLPB at very high molecular mass comigrating with the prohibitins. In control fibroblasts, HAX1 migrated predominantly as monomer while in patient samples multiple HAX1 peaks were observed at higher molecular masses comigrating with CLPB thus suggesting a longer-lasting interaction between CLPB and HAX1.
Both biallelic as well as specific monoallelic CLPB variants result in a phenotypic spectrum centered around neurodevelopmental delay, seizures, and neutropenia presumably mediated via HAX1.
The sarcoplasmic reticulum Ca-pump protein can be solubilized in monomeric form in nonionic detergents with full retention of ATPase activity. It is impossible to prove directly that coupling between ATP hydrolysis and Ca2+ transport is maintained in the soluble state, because separate compartments for Ca2+ uptake and Ca2+ discharge are required to demonstrate this, but here we provide strong indirect evidence that coupling in fact persists, in both the forward and reverse directions of the normal pump cycle. Demonstration of coupling in the forward direction makes use of the fact that the solubilized protein is structurally labile in the absence of bound Ca2+. Loss of activity accompanying ATP hydrolysis in solution is quantitatively consistent with sequential Ca2+ binding and dissociation during the hydrolysis cycle. Coupling in the reverse direction is demonstrated by the dependence of ATP synthesis on Ca2+ concentration. Although substantial differences between solubilized (monomeric) and membrane-bound protein are shown to exist, as previously reported, they do not affect the main conclusion from this work, which is that the molecular machinery for free-energy coupling in active Ca2+ transport is an inherent property of each individual catalytic polypeptide chain of the pump protein.
1.1. The binding of the ATP molecule to brain microsomal (Na+ + K+)-activated ATPase was studied by a rapid dialysis rate technique.2.2. The experiments were performed at 2° in the presence of 10 mM EDTA to minimize hydrolysis of ATP. The ionic strength was 0.073 M, pH 7.4.3.3. The specific activity of the preparations and the Na+ + K+ activity/Mg2+ activity ratio were changed by heat denaturation.4.4. The dissociation constant of the enzyme-ATP complex () was 0.12 μM.5.5. The Mg2+ requirement for binding of ATP to the enzyme, if any, was much lower than that for hydrolysis of ATP. The binding seemed to be independent of Na+.6.6. Increasing the concentration of K+ up to 3 mM led to an increase in the apparent dissociation constant of towards a maximum (0.7 μM). The effect of K+ could be described by a model involving the formation of the following complexes: , and .7.7. Proportionality was found between the ATP-binding capacity (nmoles/mg protein) and (Na+ + K+)-ATPase activity (μmoles P1 released per mg protein per h), suggesting that binding to (Na+ + K+)-ATPase was measured. The catalytic center activity based on this assumption was .
A method is described which permits the rapid simultaneous assay of numerous samples for ATPase activity. The sample is incubated with ATP and PEP, and the pyruvate liberated is enzymically coupled with NADH oxidation. The reaction is terminated and cleared after a suitable time interval by addition of sodium dodecyl sulphate (SDS) and A340 read directly.
The purification and properties of a Na+- and K+-activated ATPase from ox brain is described. The enzyme preparation is characterized by a high purity and a low content (<1%) of Mg++-stimulated ATPase.
A (Mg(2+) + Ca(2+))ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been purified from sarcoplasmic reticulum using a single step centrifugation procedure. The preparation is >95% pure by weight and contains only 25-30% of the lipid associated with the enzyme in native sarcoplasmic reticulum. The purified enzyme is unable to accumulate Ca(2+). Using a sedimentation-substitution technique, >98% of the lipid associated with the purified enzyme can be replaced by dioleoyl lecithin without grossly affecting the ATPase activity of the enzyme. The Ca(2+) pump can be restored to this dioleoyl lecithin-substituted enzyme by addition of excess sarcoplasmic reticulum lipids in the presence of cholate. Removal of the cholate by dialysis generates a system which accumulates Ca(2+) at a rate and to a level comparable to native sarcoplasmic reticulum. Significant reconstitution of the Ca(2+) pump can also be achieved using excess dioleoyl lecithin, but since the full expression of the capacity to accumulate Ca(2+) requires the presence of oxalate, these vesicles would appear to be more leaky than those reconstituted with an excess of sarcoplasmic reticulum lipids. Of about 90 lipid molecules which are associated with one molecule of ATPase in native sarcoplasmic reticulum, an average of less than one lipid molecule remains in these reconstituted systems. We have therefore achieved a fully functional Ca(2+) pump containing essentially a single protein and exogenous lipid.
Sarcoplasmic reticulum calcium adenosinetriphosphatase (Ca2+-ATPase) was solubilized to monomeric form with the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8). Equilibrium ultracentrifugation analysis indicated that this preparation is initially greater than 75% monomer, the remainder being best described as a tetramer. In the presence of substrates, this preparation has ATPase activity comparable to that of leaky sarcoplasmic reticulum vesicles. The possibility of substrate-induced oligomerization of the monomer under ATPase activity assay conditions was tested. Active enzyme centrifugation analysis demonstrated that ATPase activity sedimented with a rate which can only be attributed to a monomeric particle. The sedimentation rate was invariant over a 6-fold concentration range comparable to that used in activity assays. The portion of the protein that sediments as an oligomer when measurements are based on the movement of protein (A280) is not seen when measurements are based on the movement of activity. The data demonstrate that the monomer represents the minimal ATPase active unit of Ca2+-ATPase.