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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Diverse ATPase Proteins in Mobilomes Constitute a Large Potential Sink for Prokaryotic Host ATP

Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ~3% of the genomes of Lak phages with ~550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome infection cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire future biochemical experiments for the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The CopA2-Type P 1B -Type ATPase CcoI Serves as Central Hub for cbb 3 -Type Cytochrome Oxidase Biogenesis

Copper (Cu)-transporting P 1B -type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P 1B -type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3 -type cytochrome oxidase (cbb 3 -Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3 -Cox. CcoI and cbb 3 -Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3 -Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3 -Cox assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nucleotide- and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

Arsenite (As III ) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As III resistance mechanism in prokaryotes involves proteins encoded by thearsoperon. A central component of thearsoperon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of As III by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-boundopenstates, as well as a distinctclosedMgATP-bound state liganded to As III . X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of As III to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by As III . This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.

Science & Technology - Other Topics↗
DOE OSTI2020

“The structure of the Type III secretion system export gate with CdsO, an ATPase lever arm”

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Topobexin targets the Topoisomerase II ATPase domain for beta isoform-selective inhibition and anthracycline cardioprotection

Abstract Topoisomerase II alpha and beta (TOP2A and TOP2B) isoenzymes perform essential and non-redundant cellular functions. Anthracyclines induce their potent anti-cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. Here we describe the development of theobexclass of TOP2 inhibitors that bind to a previously unidentified druggable pocket in the TOP2 ATPase domain to act as allosteric catalytic inhibitors by locking the ATPase domain conformation with the capability of isoform-selective inhibition. Through rational drug design we have developed topobexin, which interacts with residues that differ between TOP2A and TOP2B to provide inhibition that is both selective for TOP2B and superior to dexrazoxane. Topobexin is a potent protectant against chronic anthracycline cardiotoxicity in an animal model. This demonstration of TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

Science & Technology - Other Topics↗
DOE OSTI2023

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanism of allosteric inhibition of human p97/VCP ATPase and its disease mutant by triazole inhibitors

Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been demonstrated to match the efficacy of CB-5083, an ATP-competitive inhibitor, in cellular models. However, the mechanism is not well understood. This study systematically investigates the structures of new triazole inhibitors bound to both wild-type and disease mutant forms of p97 and measures their effects on function. These inhibitors bind at the interface of the D1 and D2 domains of each p97 subunit, shifting surrounding helices and altering the loop structures near the C-terminal α2 G helix to modulate domain-domain communications. A key structural moiety of the inhibitor affects the rotameric conformations of interacting side chains, indirectly modulating the N-terminal domain conformation in p97 R155H mutant. The differential effects of inhibitor binding to wild-type and mutant p97 provide insights into drug design with enhanced specificity, particularly for oncology applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Plastic recognition and electrogenic uniport translocation of 1 st -, 2 nd -, and 3 rd -row transition and post-transition metals by primary-active transmembrane P 1B-2 -type ATPase pumps

Transmembrane P 1B -type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(II)-pumps of the P 1B-2 -type subclass, in addition to Zn 2+ , select diverse metals (Pb 2+ , Cd 2+ and Hg 2+ ) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. Yet, a comprehensive understanding of the transport of these metals, their relative translocation rates, and transport mechanism remain elusive. We developed a platform for the characterization of primary-active Zn(II)-pumps in proteoliposomes to study metal selectivity, translocation events and transport mechanism in real-time, employing a “multi-probe” approach with fluorescent sensors responsive to diverse stimuli (metals, pH and membrane potential). Together with atomic-resolution investigation of cargo selection by X-ray absorption spectroscopy (XAS), we demonstrate that Zn(II)-pumps are electrogenic uniporters that preserve the transport mechanism with 1 st -, 2 nd - and 3 rd -row transition metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Inactivation of mitochondrial complex I stimulates chloroplast ATPase in Physcomitrium patens

Abstract Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Plant Sciences↗
DOE OSTI2023

The conserved AAA ATPase PCH-2 distributes its regulation of meiotic prophase events through multiple meiotic HORMADs in C. elegans

During meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination in C. elegans by remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs in C. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2’s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Covalent labeling of the Arabidopsis plasma membrane H + ‐ ATPase reveals 3D conformational changes involving the C‐terminal regulatory domain

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C‐terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14‐3‐3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL‐MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme.

Blackburn, Matthew R. [Department of Biochemistry ↗