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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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

The hierarchical structure of organic mixed ionic–electronic conductors and its evolution in water

Polymeric organic mixed ionic–electronic conductors underpin several technologies in which their electrochemical properties are desirable. These properties, however, depend on the microstructure that develops in their aqueous operational environment. We investigated the structure of a model organic mixed ionic–electronic conductor across multiple length scales using cryogenic four-dimensional scanning transmission electron microscopy in both its dry and hydrated states. Four-dimensional scanning transmission electron microscopy allows us to identify the prevalent defects in the polymer crystalline regions and to analyse the liquid crystalline nature of the polymer. The orientation maps of the dry and hydrated polymers show that swelling-induced disorder is mostly localized in discrete regions, thereby largely preserving the liquid crystalline order. Therefore, the liquid crystalline mesostructure makes electronic transport robust to electrolyte ingress. Furthermore, this study demonstrates that cryogenic four-dimensional scanning transmission electron microscopy provides multiscale structural insights into complex, hierarchical structures such as polymeric organic mixed ionic–electronic conductors, even in their hydrated operating state.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Substrate binding plasticity revealed by Cryo-EM structures of SLC26A2

SLC26A2 is a vital solute carrier responsible for transporting essential nutritional ions, including sulfate, within the human body. Pathogenic mutations within SLC26A2 give rise to a spectrum of human diseases, ranging from lethal to mild symptoms. The molecular details regarding the versatile substrate-transporter interactions and the impact of pathogenic mutations on SLC26A2 transporter function remain unclear. Here, using cryo-electron microscopy, we determine three high-resolution structures of SLC26A2 in complexes with different substrates. These structures unveil valuable insights, including the distinct features of the homodimer assembly, the dynamic nature of substrate binding, and the potential ramifications of pathogenic mutations. This structural-functional information regarding SLC26A2 will advance our understanding of cellular sulfate transport mechanisms and provide foundations for future therapeutic development against various human diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found unique structural variabilities in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure’s impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improving resolution and resolvability of single-particle cryoEM structures using Gaussian mixture models

Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here, we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation, and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Human TRPV1 structure and inhibition by the analgesic SB-366791

Pain therapy has remained conceptually stagnant since the opioid crisis, which highlighted the dangers of treating pain with opioids. An alternative addiction-free strategy to conventional painkiller-based treatment is targeting receptors at the origin of the pain pathway, such as transient receptor potential (TRP) ion channels. Thus, a founding member of the vanilloid subfamily of TRP channels, TRPV1, represents one of the most sought-after pain therapy targets. The need for selective TRPV1 inhibitors extends beyond pain treatment, to other diseases associated with this channel, including psychiatric disorders. Here we report the cryo-electron microscopy structures of human TRPV1 in the apo state and in complex with the TRPV1-specific nanomolar-affinity analgesic antagonist SB-366791. SB-366791 binds to the vanilloid site and acts as an allosteric hTRPV1 inhibitor. SB-366791 binding site is supported by mutagenesis combined with electrophysiological recordings and can be further explored to design new drugs targeting TRPV1 in disease conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular pathway and structural mechanism of human oncochannel TRPV6 inhibition by the phytocannabinoid tetrahydrocannabivarin

The calcium-selective oncochannel TRPV6 is an important driver of cell proliferation in human cancers. Despite increasing interest of pharmacological research in developing synthetic inhibitors of TRPV6, natural compounds acting at this channel have been largely neglected. On the other hand, pharmacokinetics of natural small-molecule antagonists optimized by nature throughout evolution endows these compounds with a medicinal potential to serve as potent and safe next-generation anti-cancer drugs. Here we report the structure of human TRPV6 in complex with tetrahydrocannabivarin (THCV), a natural cannabinoid inhibitor extracted from Cannabis sativa . We use cryo-electron microscopy combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to identify THCV binding sites in the portals that connect the membrane environment surrounding the protein to the central cavity of the channel pore and to characterize the allosteric mechanism of TRPV6 inhibition. We also propose the molecular pathway taken by THCV to reach its binding site. Our study provides a foundation for the development of new TRPV6-targeting drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The CNK–HYP scaffolding complex promotes RAF activation by enhancing KSR–MEK interaction

The RAS–MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1–BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR–MEK interaction, which in turn enhances RAF–KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR–MEK–CNK–HYP complex reveals a ring-like arrangement of the CNK–HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR–MEK interaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cryo-EM structures of PP2A:B55–FAM122A and PP2A:B55–ARPP19

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP19 and FAM122A. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding

The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Optical interference for the guidance of cryogenic focused ion beam milling beyond the axial diffraction limit

Cryogenic focused ion beam (Cryo-FIB) milling has become a standard step in the cryogenic electron tomography (Cryo-ET) workflow and is required to thin cells to electron-semitransparency. However, this destructive process removes the vast majority of the cellular material and raises a critical question: what thin section should be preserved for Cryo-ET analysis? Using a tri-coincident cryogenic FIB-SEM-LM system, we identify an interferometric optical response that can be used for targeting lamella production to fluorescently labeled structures with accuracy beyond the diffraction limit. Here we demonstrate this approach using synthetic samples of fluorescent beads embedded in micron-scale droplets of amorphous ice. We then apply the approach to capture virions inside host cells. Successful targeting is confirmed by Cryo-ET revealing clusters of virions in intracellular vesicles. The method does not require any fluorescent fiducials or axial registration and can be performed on any fluorescently labeled structure that is visible in widefield fluorescence microscopy.

Cryoelectron microscopy↗
DOE OSTI2023

Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis

Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cell membranes. In eukaryotes, two highly homologous enzymes, cholinephosphotransferase-1 (CHPT1) and choline/ethanolamine phosphotransferase-1 (CEPT1) catalyze the final step of de novo PC synthesis. CHPT1/CEPT1 joins two substrates, cytidine diphosphate-choline (CDP-choline) and diacylglycerol (DAG), to produce PC, and Mg 2+ is required for the reaction. However, mechanisms of substrate recognition and catalysis remain unresolved. Here we report structures of a CHPT1 from Xenopus laevis (xlCHPT1) determined by cryo-electron microscopy to an overall resolution of ~3.2 Å. xlCHPT1 forms a homodimer, and each protomer has 10 transmembrane helices (TMs). The first 6 TMs carve out a cone-shaped enclosure in the membrane in which the catalysis occurs. The enclosure opens to the cytosolic side, where a CDP-choline and two Mg 2+ are coordinated. The structures identify a catalytic site unique to eukaryotic CHPT1/CEPT1 and suggest an entryway for DAG. The structures also reveal an internal pseudo two-fold symmetry between TM3-6 and TM7-10, and suggest that CHPT1/CEPT1 may have evolved from their distant prokaryotic ancestors through gene duplication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Kainate receptor channel opening and gating mechanism

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism. Although structures of kainate receptor domains and subunit assemblies are available, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis for $\mathrm{APE1}$ processing $\mathrm{DNA}$ damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3-6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural polymorphism of amyloid fibrils in ATTR amyloidosis revealed by cryo-electron microscopy

ATTR amyloidosis is caused by the deposition of transthyretin in the form of amyloid fibrils in virtually every organ of the body, including the heart. This systemic deposition leads to a phenotypic variability that has not been molecularly explained yet. In brain amyloid conditions, previous studies suggest an association between clinical phenotype and the molecular structures of their amyloid fibrils. Here we investigate whether there is such an association in ATTRv amyloidosis patients carrying the mutation I84S. Using cryo-electron microscopy, we determined the structures of cardiac fibrils extracted from three ATTR amyloidosis patients carrying the ATTRv-I84S mutation, associated with a consistent clinical phenotype. We found that in each ATTRv-I84S patient, the cardiac fibrils exhibited different local conformations, and these variations can co-exist within the same fibril. Our finding suggests that one amyloid disease may associate with multiple fibril structures in systemic amyloidoses, calling for further studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis for SHOC2 modulation of RAS signalling

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase RAF remains limited. Recent structures of inactive RAF monomer and active RAF dimer bound to 14-3-3 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family.

60 APPLIED LIFE SCIENCES↗