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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 667 records · Page 37
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome

Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N 1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of agonist specificity of α 1A -adrenergic receptor

α 1 -adrenergic receptors (α 1 -ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α 1 subtypes has limited our understanding of the physiological roles of different α 1 -AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α 1A -AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α 1A -AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α 1A -AR and α 1B -AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α 1A -AR mutants that are not responsive to A61603, and α 1B -AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α 1 -ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for DNA proofreading

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved ‘bolt-action’ mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin’s potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis for self-discrimination by neoantigen-specific TCRs

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-D b /Hsf2 p.K72N 68-76 , as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-D b binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The structural basis for 2′−5′/3′−5′-cGAMP synthesis by cGAS

Abstract cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis of tRNA recognition by the widespread OB fold

The widespread oligonucleotide/oligosaccharide-binding (OB)-fold recognizes diverse substrates from sugars to nucleic acids and proteins, and plays key roles in genome maintenance, transcription, translation, and tRNA metabolism. OB-containing bacterial Trbp and yeast Arc1p proteins are thought to recognize the tRNA elbow or anticodon regions. Here we report a 2.6 Å co-crystal structure of Aquifex aeolicus Trbp111 bound to tRNA Ile , which reveals that Trbp recognizes tRNAs solely by capturing their 3' ends. Structural, mutational, and biophysical analyses show that the Trbp/EMAPII-like OB fold precisely recognizes the single-stranded structure, 3' terminal location, and specific sequence of the 3' CA dinucleotide — a universal feature of mature tRNAs. Arc1p supplements its OB – tRNA 3' end interaction with additional contacts that involve an adjacent basic region and the tRNA body. This study uncovers a previously unrecognized mode of tRNA recognition by an ancient protein fold, and provides insights into protein-mediated tRNA aminoacylation, folding, localization, trafficking, and piracy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis for C-degron selectivity across KLHDCX family E3 ubiquitin ligases

Abstract Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate’s C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10’s recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.

Science & Technology - Other Topics↗
DOE OSTI2024

Mechanistic basis of atypical TERT promoter mutations

Non-coding mutations in the TERT promoter (TERTp), typically at one of two bases -124 and -146 bp upstream of the start codon, are among the most prevalent driver mutations in human cancer. Several additional recurrent TERTp mutations have been reported but their functions and origins remain largely unexplained. Here, we show that atypical TERTp mutations arise secondary to canonical TERTp mutations in a two-step process. Canonical TERTp mutations create de novo binding sites for ETS family transcription factors that induce favourable conditions for DNA damage formation by UV light, thus creating a hotspot effect but only after a first mutational hit. In agreement, atypical TERTp mutations co-occur with canonical driver mutations in large cancer cohorts and arise subclonally specifically on the TERTp driver mutant chromosome homolog of melanoma cells treated with UV light in vitro. Our study gives an in-depth view of TERTp mutations in cancer and provides a mechanistic explanation for atypical TERTp mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates

The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural basis for catalysis by human lipoyl synthase

Abstract Lipoic acid is an essential cofactor in five mitochondrial multiprotein complexes. In each complex, it is tethered in an amide linkage to the side chain of a conserved lysyl residue on a lipoyl carrier protein or lipoyl domain to afford the lipoyl cofactor. Lipoyl synthase catalyzes the last step in the biosynthesis of the lipoyl cofactor, the addition of two sulfur atoms to carbons 6 and 8 of an octanoyllysyl residue of the H protein, the lipoyl carrier protein of the glycine cleavage system. Lipoyl synthase, a member of the radical S-adenosylmethionine superfamily, contains two [Fe 4 S 4 ] clusters, one of which is sacrificed during catalysis to supply the appended sulfur atoms. Herein, we use X-ray crystallography to characterize several stages in lipoyl synthase catalysis and present a structure of an intermediate wherein the enzyme is cross-linked to the H protein substrate through a 6-mercaptooctanoyl ligand to a [Fe 3 S 4 ] cluster.

Science & Technology - Other Topics↗
DOE OSTI2025

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

Ashraf, Khuram U. [Rutgers University, Newark, NJ ↗
DOE OSTI2026

Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

Small-conductance (K Ca 2.2) and intermediate-conductance (K Ca 3.1) Ca 2+ -activated K + channels are gated by a Ca 2+ -calmodulin dependent mechanism. NS309 potentiates the activity of both K Ca 2.2 and K Ca 3.1, while rimtuzalcap selectively activates K Ca 2.2. Rimtuzalcap has been used in clinical trials for the treatment of spinocerebellar ataxia and essential tremor. We report cryo-electron microscopy structures of NS309-bound K Ca 2.2 and K Ca 3.1, in addition to structures of rimtuzalcap-bound K Ca 2.2 and mutant K Ca 3.1_R355K. The different conformations of calmodulin and the cytoplasmic HC helices in the two channels underlie the subtype-selectivity of rimtuzalcap for K Ca 2.2. NS309 binds to pre-existing pockets in both channels, while the bulkier rimtuzalcap binds in an induced-fit pocket in K Ca 2.2 requiring conformational changes. In K Ca 2.2, calmodulin’s N-lobes are sufficiently far apart to enable conformational changes to accommodate either NS309 or rimtuzalcap. In K Ca 3.1, calmodulin’s N-lobes are closer to each other and constrained by K Ca 3.1’s HC helices, which allows binding of NS309 but not rimtuzalcap. Replacement of arginine-355 in K Ca 3.1’s HB helix with lysine (K Ca 3.1_R355K) allows the binding of rimtuzalcap and renders the mutant channel sensitive to rimtuzalcap. These structures provide a framework for structure-based drug design targeting K Ca 2.2 channels.

Nam, Young-Woo [Chapman Univ., Irvine, CA (United ↗
DOE OSTI2026

Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase

Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O 2 surrogate nitric oxide imply O 2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O 2 is the sole oxygen source in alcohol products, as shown by 18 O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.

de Munnik, Mariska↗
DOE OSTI2022

Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

60 APPLIED LIFE SCIENCES↗