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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 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural basis of broad HIV neutralization by a vaccine-induced cow antibody

Potent broadly neutralizing antibodies (bnAbs) to HIV have been very challenging to elicit by vaccination in wild-type animals. Here, by x-ray crystallography, cryo–electron microscopy, and site-directed mutagenesis, we structurally and functionally elucidate the mode of binding of a potent bnAb (NC-Cow1) elicited in cows by immunization with the HIV envelope (Env) trimer BG505 SOSIP.664. The exceptionally long (60 residues) third complementarity-determining region of the heavy chain (CDR H3) of NC-Cow1 forms a mini domain (knob) on an extended stalk that navigates through the dense glycan shield on Env to target a small footprint on the gp120 CD4 receptor binding site with no contact of the other CDRs to the rest of the Env trimer. These findings illustrate, in molecular detail, how an unusual vaccine-induced cow bnAb to HIV Env can neutralize with high potency and breadth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Dynamic behavior of chemically tunable mechano-responsive hydrogels

Here, using theory and simulation, we model the mechanical behavior of gels that encompass loops and dangling chain ends. If the loops remain folded and dangling ends are chemically inert, then these topological features just serve as defects. If, however, the loops unfold to expose the hidden (“cryptic”) binding sites and the ends of the dangling chains are reactive, these moieties can form bonds that improve the gel's mechanical properties. For gels with a lower critical solubility temperature (LCST), we systematically switch on the possible unfolding and binding events. To quantify the resulting effects, we derive equations for the gel's equilibrium and dynamic elastic moduli. We also use a finite element approach to simulate the gel's response to deformation and validate the analytic calculations. Herein, we show that the equilibrium moduli are highly sensitive to the presence of unfolding and binding transitions. The dynamical moduli are sensitive not only to these structural changes, but also to the frequency of deformation. For example, when reactive ends bind to exposed cryptic sites at T = 29 °C and relatively high frequency, the storage shear modulus is 119% greater than the corresponding equilibrium value, while the storage Young's modulus is 109% greater than at equilibrium. These findings provide guidelines for tuning the chemical reactivity of loops and dangling ends and the frequency of deformation to tailor the mechano-responsive behavior of polymer networks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor’s conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1,2,3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure–function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Additionally, newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure–function relationships in ribozymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Ni( II )-binding affinity of Cc NikZ-II and its homologs: the role of the HH -prong and variable loop revealed by structural and mutational studies

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein–coupled receptors. Here, we report the cryo–electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ–adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101–specific signaling events in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The histone H3-H4 tetramer is a copper reductase enzyme

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu 2+ ) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu 2+ and catalyzes its reduction to Cu 1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu 1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae . The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu 1+ ions in eukaryotes.

Attar, Narsis↗
DOE OSTI2020

Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family

The fundamental and assorted roles of β-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. Herein, the selectivity and modes of action of a glycoside hydrolase family active on β-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/β)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical β-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of β-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of β-1,3-glucans, which can be exploited for biotechnological applications.

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

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. In this work, five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Hit Expansion of a Noncovalent SARS-CoV-2 Main Protease Inhibitor

Inhibition of the SARS-CoV-2 main protease (M pro ) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of M pro . Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 μM. Among these, four compounds with IC 50 values around 1 μM were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Room-temperature X-ray structures of co-crystallized protein–inhibitor complexes were determined up to 1.9 Å resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for M pro inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B 12

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. Finally, the data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

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

Solution Structures of Europium Terpyridyl Complexes with Nitrate and Triflate Counterions in Acetonitrile

Lanthanide–ligand complexes are key components of technological applications, and their properties depend on their structures in the solution phase, which are challenging to resolve experimentally or computationally. The coordination structure of the Eu 3+ ion in different coordination environments in acetonitrile is examined using ab initio molecular dynamics (AIMD) simulations and extended X-ray absorption fine structure (EXAFS) spectroscopy. AIMD simulations are conducted for the solvated Eu 3+ ion in acetonitrile, both with or without a terpyridyl ligand, and in the presence of either triflate or nitrate counterions. EXAFS spectra are calculated directly from AIMD simulations and then compared to experimentally measured EXAFS spectra. In acetonitrile solution, both nitrate and triflate anions are shown to coordinate directly to the Eu 3+ ion forming either ten- or eight-coordinate solvent complexes where the counterions are binding as bidentate or monodentate structures, respectively. Coordination of a terpyridyl ligand to the Eu 3+ ion limits the available binding sites for the solvent and anions. In certain cases, the terpyridyl ligand excludes any solvent binding and limits the number of coordinated anions. The solution structure of the Eu-terpyridyl complex with nitrate counterions is shown to have a similar arrangement of Eu 3+ coordinating molecules as the crystal structure. Finally, this study illustrates how a combination of AIMD and EXAFS can be used to determine how ligands, solvent, and counterions coordinate with the lanthanide ions in solution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

EF-Hand Battle Royale: Hetero-ion Complexation in Lanmodulin

The lanmodulin (LanM) protein has emerged as an effective means for rare earth element (REE) extraction and separation from complex feedstocks without the use of organic solvents. Whereas the binding of LanM to individual REEs has been well characterized, little is known about the thermodynamics of mixed metal binding complexes (i.e., heterogeneous ion complexes), which limits the ability to accurately predict separation performance for a given metal ion mixture. In this paper, we employ the law of mass action to establish a theory of perfect cooperativity for LanM-REE complexation at the two highest-affinity binding sites. The theory is then used to derive an equation that explains the nonintuitive REE binding behavior of LanM, where separation factors for binary pairs of ions vary widely based on the ratio of ions in the aqueous phase, a phenomenon that is distinct from single-ion-binding chemical chelators. We then experimentally validate this theory and perform the first quantitative characterization of LanM complexation with heterogeneous ion pairs using resin-immobilized LanM. Importantly, the resulting homogeneous and heterogeneous constants enable accurate prediction of the equilibrium state of LanM in the presence of mixtures of up to 10 REEs, confirming that the perfect cooperativity model is an accurate mechanistic description of REE complexation by LanM. We further employ the model to simulate separation performance over a range of homogeneous and heterogeneous binding constants, revealing important insights into how mixed binding differentially impacts REE separations based on the relative positioning of the ion pairs within the lanthanide series. In addition to informing REE separation process optimization, these results provide mathematical and experimental insight into competition dynamics in other ubiquitous and medically relevant, cooperative binding proteins, such as calmodulin.

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

Conformational heterogeneity in the dGsw purine riboswitch: role of Mg²⁺ and 2’-dG in aptamer folding

Recent advancements in RNA structural biology have focused on unraveling the complexities of non-coding mRNA elements like riboswitches. These cis-acting regulatory regions undergo structural changes in response to specific cellular metabolites, leading to up or downregulation of downstream genes. The purine riboswitch family regulates many prokaryotic genes involved in purine degradation and biosynthesis. They feature an aptamer domain organized around a 3-way helical junction, where ligand encapsulation occurs at the junctional core. In our study, we chemically probed the aptamer domain of the 2’-dG-sensing purine riboswitch from Mesoplasma florum (dGsw) under various solution conditions to understand how Mg²⁺ and 2’-dG influence riboswitch folding. Here, we find that efficient 2’-dG binding strongly depends on Mg²⁺, indicating that Mg²⁺ is essential for priming dGsw for ligand interactions. We identified a previously undescribed sequence in the 5’ tail of dGsw that is complementary to a conserved helix. The inclusion of this region in a construct led to intramolecular competition between the alternate helix, Palt, and P1. Mutational analysis confirmed that 5’ flanking end of the aptamer domain forms an alternate helix in the absence of ligand. Molecular dynamics simulations revealed that this alternative conformation is stable. This helix may, therefore, facilitate the formation of an anti-terminator helix by opening the 3-way junction surrounding the 2’-dG binding site. Our study further establishes the importance of a closed terminal P1 helix conformation for metabolite binding and suggests that the delicate interplay between P1 and Palt may fine-tune downstream gene regulation. These insights offer a new perspective on riboswitch structure and enhance our understanding of the role that a conformational ensemble plays in riboswitch activity and regulation.

Biochemistry & Molecular Biology↗