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Results for “ATP hydrolysis”

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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31 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Transforming an ATP-dependent enzyme into a dissipative, self-assembling system

Nucleoside triphosphate (NTP)-dependent protein assemblies such as microtubules and actin filaments have inspired the development of diverse chemically fueled molecular machines and active materials but their functional sophistication has yet to be matched by design. Given this challenge, we asked whether it is possible to transform a natural adenosine 5'-triphosphate (ATP)-dependent enzyme into a dissipative self-assembling system, thereby altering the structural and functional mode in which chemical energy is used. Furthermore we report that FtsH (filamentous temperature-sensitive protease H), a hexameric ATPase involved in membrane protein degradation, can be readily engineered to form one-dimensional helical nanotubes. FtsH nanotubes require constant energy input to maintain their integrity and degrade over time with the concomitant hydrolysis of ATP, analogous to natural NTP-dependent cytoskeletal assemblies. Yet, in contrast to natural dissipative systems, ATP hydrolysis is catalyzed by free FtsH protomers and FtsH nanotubes serve to conserve ATP, leading to transient assemblies whose lifetimes can be tuned from days to minutes through the inclusion of external ATPases in solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Mechanical coupling in the nitrogenase complex

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188Ser) are important for communication between the two halves.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Nitrogenase Bioelectrocatalysis: ATP-Independent Ammonia Production Using a Redox Polymer/MoFe Protein System

Nitrogenase is the only biological catalyst that is understood to be able to convert nitrogen gas to ammonia. In microorganisms, the MoFe catalytic protein of nitrogenase is reduced by a transient Fe protein binding and separate hydrolysis of ATP. Yet, the requirement of 16 ATP molecules by the Fe protein for the 8 electron transfer is an energy-intense caveat to the enzymatic synthesis of NH 3 and is challenging from an electrochemical perspective. Thus, we report the redox polymer-based ATP-free mediated electron-transfer system of MoFe nitrogenase using cobaltocene-functionalized poly(allylamine) (Cc-PAA), which is able to reduce the MoFe nitrogenase directly with a low redox potential of -0.58 V vs SHE. An efficient immobilization of MoFe nitrogenase via Cc-PAA allowed for the bioelectrocatalytic reduction of N 3 – , NO 2 – , and N 2 to NH 3 . Bulk bioelectrosynthetic experiments produced 7 ± 2 and 30 ± 5 nmol of NH 3 from NO 2 – and N 3 – reduction for 30 min, respectively. In addition, biosynthetic N 2 reduction to NH 3 was confirmed by 15 N 2 labeling experiments with NMR analysis. This mediated electron-transfer approach of the immobilized nitrogenase using the Cc-PAA redox polymer provides a valuable technological basis for scale-up and industrial uses in the future of bioelectrosynthesis.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2020

Divergent Members of the Nitrogenase Superfamily: Tetrapyrrole Biosynthesis and Beyond

The nitrogenase superfamily constitutes a large and diverse ensemble of two–component metalloenzymes. These systems couple the hydrolysis of ATP to the reduction of disparate substrates from diatomic gases (Mo and alternative nitrogenases) to photosynthetic pigments (protochlorophyllide and chlorophyllide oxidoreductases). Only very recently have the activities of the highly divergent and paraphyletic Group IV nitrogenases begun to be uncovered. In conclusion, this review highlights the first characterized member of this group, which was found to catalyze an unprecedented reaction in the coenzyme F430 biosynthetic pathway, and the catalytic potential of a superfamily that has yet to be fully explored.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Mechanistic Insights into Dinitrogen Reduction to Ammonia in Light-Controlled Nanocrystal:Nitrogenase Complexes

Developing systems that can efficiently capture photon energy and convert this energy into fuels and chemicals requires understanding how to assemble molecular components with diverse functions into complete systems possessing selectivity and efficiency in directing charge carriers to catalytic reactions. There are many challenges to achieving this goal. One promising approach is the development of hybrid systems that combine semiconductor nanocrystals (NCs) for light capture and enzymes as efficient catalysts. Such biohybrid systems capitalize on the tunable electronic and optical properties of NCs while leveraging the unmatched specificity and efficiency of enzymes in catalyzing chemical reactions, thereby offering opportunities to surpass the limitations of each component alone. Here, we focus on recent progress in developing a biohybrid system that combines CdS NCs for photon capture with the enzyme nitrogenase to accomplish light-driven dinitrogen (N 2 ) reduction to ammonia (NH 3 ). Integrating light-harvesting materials with biological catalysts requires a deep understanding of NC properties, protein stability, and electron transfer (ET), making it an inherently multidisciplinary problem. The reduction of N 2 to NH 3 is a challenging reaction, with a high demand in both agriculture and industrial chemical production. This reaction is intrinsically energy intensive, due to the need to activate the N≡N triple bond. The current standard industrial approach to N 2 reduction, the Haber−Bosch reaction, obtains the necessary energy input from fossil fuels, whereas biological systems capable of N 2 reduction utilize the hydrolysis of ATP as their energy source. Replacing these costly, energy-intensive inputs with renewable light energy represents a critical step toward sustainable NH 3 production. Recent progress has demonstrated that semiconductor CdS NCs can be coupled to the catalytic component of nitrogenase, the MoFe protein, to form a biohybrid CdS NC:MoFe protein complex, enabling light-driven N 2 reduction rather than energy input from fossil fuels or ATP. This illustrates how inorganic NCs can functionally replace the natural Fe protein partner, yielding a biohybrid catalyst that enables controlled electron delivery and provides not only light-driven NH 3 production but also new approaches for probing enzyme catalytic function. The CdS NC:MoFe protein biohybrid system enables light-initiated electron delivery at ambient temperature, as well as temperatures below freezing, allowing for stabilization and spectroscopic characterization of key reaction intermediates. These findings highlight how photochemical biohybrids can serve as both functional catalysts and mechanistic probes. Beyond studies of the nitrogenase mechanism, studies of the CdS NC:MoFe system reveal how variables such as NC size, electrostatic binding interactions, and sacrificial electron donors (SEDs) govern complex stability, charge transfer efficiency, and catalytic performance. In addition, studies of nitrogenase and the high activation barrier for N 2 reduction are enabling investigations of new and interesting questions regarding the properties and limitations of NC biocatalysis. In this Account, we describe the key features of CdS NC:MoFe protein biohybrids and the parameters for optimal light-driven N 2 reduction, and how controlling ET with light illuminates the path to new insights into the nitrogenase mechanism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR

Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an α 2 β 2 heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.

Computational biophysics↗
DOE OSTI2021

Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex

The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA + ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3–REV7 binary and fused SHLD2–SHLD3–REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2–SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2–SHLD3–REV7–TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Active conformation of the p97-p47 unfoldase complex

The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a “hand-over-hand” mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures

Abstract Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg 2+ , and, more importantly, a putative third (site C) Mg 2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg 2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

Science & Technology - Other Topics↗