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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 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural basis for aminoacylation of cellular modified tRNALys3 by human lysyl-tRNA synthetase

Abstract The average eukaryotic transfer ribonucleic acid (tRNA) contains 13 post-transcriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully modified cellular tRNALys3 by human lysyl-tRNA synthetase (h-LysRS). The tRNALys3 anticodon loop modifications S34 (mcm5s2U) and R37 (ms2t6A) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNALys3 are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNALys3 aminoacylation are structurally ordered; docking of the 3′-CCA end in the active site cannot proceed until the lysyl–adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS–tRNALys3 complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium toward the 3′-CCA end “docked” conformation and allosterically increases h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.

Devarkar, Swapnil C. (ORCID:000000029271243X)↗
NASA NTRS2004

The Thiamine-Pyrophosphate-Motif

Thiamin pyrophosphate (TPP), a derivative of vitamin B1, is a cofactor for enzymes performing catalysis in pathways of energy production including the well known decarboxylation of a-keto acid dehydrogenases followed by transketolation. TPP-dependent enzymes constitute a structurally and functionally diverse group exhibiting multimeric subunit organization, multiple domains and two chemically equivalent catalytic centers. Annotation of functional TPP-dependcnt enzymes, therefore, has not been trivial due to low sequence similarity related to this complex organization. Our approach to analysis of structures of known TPP-dependent enzymes reveals for the first time features common to this group, which we have termed the TPP-motif. The TPP-motif consists of specific spatial arrangements of structural elements and their specific contacts to provide for a flip-flop, or alternate site, enzymatic mechanism of action. Analysis of structural elements entrained in the flip-flop action displayed by TPP-dependent enzymes reveals a novel definition of the common amino acid sequences. These sequences allow for annotation of TPP-dependent enzymes, thus advancing functional proteomics. Further details of three-dimensional structures of TPP-dependent enzymes will be discussed.

Ciszak, Ewa↗
DOE OSTI2024

Structural basis for intermodular communication in assembly-line polyketide biosynthesis

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. Here, to investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

PARP1-HPF1 structure and dynamics on nicked DNA suggest a mechanism for acute and localized ADP-ribosylation

PARP1 detection of DNA strand breaks allosterically leads to PARP1 synthesis of poly(ADP-ribose) modifications that signal DNA damage. HPF1 engages activated PARP1 to control modification site selection. Understanding of the mechanism of DNA break detection and catalytic activation is incomplete, due largely to limited structural information for full-length PARP1. Here, single-particle cryo-EM provides views of the full complement of PARP1 domains engaging a DNA single-strand break in the presence of HPF1 and a fragment of binding partner Timeless. Cryo-EM, single-molecule DNA dynamics, and small-angle X-ray scattering analysis indicate that PARP1 remains dynamic even when the multi-domain structure is organized on a DNA break, with the minimal catalytic region displaying high mobility relative to domains engaging damage. We propose that the organization of PARP1 domains on a DNA break releases a tethered, constitutively active catalytic region to modify molecules in a radius surrounding the DNA break site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex

Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1–EXT2 (EXT1–2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. Furthermore, the two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

The Thiamin Pyrophosphate-Motif

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits, two catalytic centers, common amino acid sequence, and specific contacts to provide a flip-flop, or alternate site, mechanism of action. Each catalytic center [PP:PYR] is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and aminopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core [PP:PYR]* within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GX@&(G)@XXGQ, and GDGX25-30 within the PP- domain, and the E&(G)@XXG@ within the PYR-domain, where Q, corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Dominiak, Paulina M.↗
NASA NTRS2003

The Thiamin Pyrophosphate-Motif

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits and two catalytic centers. Each catalytic center (PP:PYR) is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and amhopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core (PP:PYR)(sub 2) within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GXPhiX(sub 4)(G)PhiXXGQ and GDGX(sub 25-30)NN in the PP-domain, and the EX(sub 4)(G)PhiXXGPhi in the PYR-domain, where Phi corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Dominiak, P.↗
NASA NTRS2003

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-Dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

Thiamin pyrophosphate (TPP), the biologically active form of vitamin BI, is a cofactor of enzymes catalyzing reactions involving the cleavage of a carbon-carbon bond adjacent to an oxo group. TPP-dependent enzymes show a common mechanism of TPP activation by: (1) forming the ionic N-H...O(sup -) hydrogen bonding between the N1' atom of the aminopirymidine ring of the coenzyme and intrinsic gamma-carboxylate group of glutamate and (2) imposing an "active" V-conformation that brings the N4' atom of the aminopirymidine to the distance required for the intramolecular C-H.. .N hydrogen bonding with the thiazolium C2 atom. Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1' atom is strictly coordinated with the deprotonation of the 4' -amino group and eventually abstraction of the proton from C2. The human pyruvate dehydrogenase Elp, component of human pyruvate dehydrogenase complex, catalyzes the irreversible decarboxylation of the pyruvate followed by the reductive acetylation of the lipoyl group of dihydrolipoyl acyltransferase. Elp is alpha(sub 2)beta(sub2)-heterotetrameric with a molecular mass of I54 kDa, which has two catalytic sites, each providing TPP and magnesium ion as cofactors and each formed on the interface between the PP and PYR domains. The dynamic nonequivalence of two otherwise chemically equivalent catalytic sites has been observed and the flip-flop mechanism was suggested, according to which two active sites affect each other and in which different steps of the catalytic reaction are performed in each of the sites at any given moment. Based on specific futures of human pyruvate dehydrogenase including rigid and flexible connections between domains that bind the cofactor we propose a mechanistic model for the flip-flop action of this enzyme. We postulate that the dynamic protein environment drives the exchange of tautomers in the 4' -aminopyrimidine ring of the cofactor through a concerted shuttl-like motion of tightly connected domains. The dynamic exchange of those tautomers, in turns, is required during the reactions of pyruvate decarboxylation and reductive acetylation of lipoamide. Thus the shuttle-like motion of the domains is coordinated with the reactions of decarboxylation and acetylation, which are carried out in each of the cofactor sites resulting in a flip-flop action of the enzyme. The structure-derived mechanism of action of human pyruvate dehydrogenase may be likely common for other TPP-dependent enzymes.

Dominiak, Paulina↗
DOE OSTI2023

Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K D values in the low millimolar range. Since these K D values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Fermentation Practices Select for Thermostable Endolysins in Phages

Endolysins are produced by (bacterio)phages and play a crucial role in degrading the bacterial cell wall and the subsequent release of new phage progeny. These lytic enzymes exhibit a remarkable diversity, often occurring in a multimodular form that combines different catalytic and cell wall-binding domains, even in phages infecting the same species. Yet, our current understanding lacks insight into how environmental factors and ecological niches may have influenced the evolution of these enzymes. In this study, we focused on phages infecting Streptococcus thermophilus, as this bacterial species has a well-defined and narrow ecological niche, namely, dairy fermentation. Among the endolysins found in phages targeting this species, we observed limited diversity, with a singular structural type dominating in most of identified S. thermophilus phages. Within this prevailing endolysin type, we discovered a novel and highly conserved calcium-binding motif. This motif proved to be crucial for the stability and activity of the enzyme at elevated temperatures. Ultimately, we demonstrated its positive selection within the host's environmental conditions, particularly under the temperature profiles encountered in the production of yogurt, mozzarella, and hard cheeses that rely on S. thermophilus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2022

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2002

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

NASA Discipline Cell Biology↗
DOE OSTI2025

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗
DOE OSTI2020

3D domain swapping in the TIM barrel of the α subunit of Streptococcus pneumoniae tryptophan synthase

Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

3D domain swapping↗
DOE OSTI2020

Structural basis for Ca2+-dependent activation of a plant metacaspase

Abstract Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca 2+ for their activation and substrate processing. However, the Ca 2+ -dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana ( At MC4) that modulates Ca 2+ -dependent, damage-induced plant immune defense. The At MC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of At MC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca 2+ . Our analysis provides insight into the Ca 2+ -dependent activation of At MC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

PII interactions with the acetyl-CoA carboxylase subunits BADC and BCCP co-regulate lipid and nitrogen metabolism in Arabidopsis

In plants, the initiation of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACCase), which produces malonyl-CoA. The heteromeric form of ACCase (htACCase) is a holoenzyme consisting of biotin carboxylase and carboxyltransferase sub-complexes, both of which are subject to extensive regulation. Biotin carboxylase activity is controlled in part by the presence of the catalytic biotin carboxyl carrier proteins (BCCP1/2) and/or the non-catalytic, non-biotinylated, biotin/lipoyl attachment domain-containing proteins (BADC1/2/3) that associate with backbone biotin carboxylase (BC) protein. However, the mechanisms regulating BADC and BCCP interactions with BC and, consequently, ACCase activity in planta , remain unclear. Here, we demonstrate that the Arabidopsis ( Arabidopsis thaliana ) regulatory protein PII modulates htACCase activity through independent interactions with BADC and BCCP proteins in a selective manner. Analysis of badc1 badc2 and badc1 badc3 mutant lines and the respective pii triple mutants revealed that changes in seed oil and protein accumulation of badc double mutants are PII/nitrogen dependent. Absolute quantification of htACCase subunits and PII in developing seeds suggests that Arabidopsis exerts tight regulation over individual protein stoichiometry to balance oil and protein accumulation. The effects on vegetative and seed development indicate that PII and BADC proteins have distinct yet overlapping roles in regulating plant metabolism.

Garneau, Matthew G. [Washington State Univ., Pullm↗