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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 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Peripheral positions encode transport specificity in the small multidrug resistance exporters

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Science & Technology - Other Topics↗
DOE OSTI2020

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA–Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and Function of the Zinc Binding Protein ZrgA from Vibrio cholerae

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISS OMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISS OMP . The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISS OMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Bacterial hemophilin homologs and their specific type eleven secretor proteins have conserved roles in heme capture and are diversifying as a family

Cellular life relies on enzymes that require metals, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogens Neisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii, and Haemophilus influenzae. Experimentally verified T11SS-dependent cargo include transferrin-binding protein B (TbpB), the hemophilin homologs heme receptor protein C (HrpC), hemophilin A (HphA), the immune evasion protein factor-H binding protein (fHbp), and the host symbiosis factor nematode intestinal localization protein C (NilC). Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explored the ligand binding ability of those hemophilin cargo homologs. In vivo expression in Escherichia coli of hemophilin homologs revealed that each is secreted in a specific manner by its cognate T11SS protein. Sequence analysis and structural modeling suggest that all hemophilin homologs share an N-terminal ligand-binding domain with the same topology as the ligand-binding domains of the Haemophilus haemolyticus heme binding protein (Hpl) and HphA. We term this signature feature of this group of proteins the hemophilin ligand-binding domain. Network analysis of hemophilin homologs revealed five subclusters and representatives from four of these showed variable heme-binding activities, which, combined with sequence-structure variation, suggests that hemophilins are diversifying in function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Abstract Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis , here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Pumping Iron: A Multi-omics Analysis of Two Extremophilic Algae Reveals Iron Economy Management

Marine algae are responsible for half of the world's primary productivity, but this critical carbonsink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, isremarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A relatedspecies, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced highquality genome assemblies and transcriptomes for both species to serve as a foundation for acomparative multi-omics analysis. We identified a host of iron-uptake proteins in both species,including a massive expansion of transferrins and a novel family of siderophore-iron uptakeproteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large ironreservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reducedinvestment in the photosynthetic apparatus coupled with remodeling of antenna proteins bydramatic iron-deficiency induction of TIDI1, a light harvesting complex protein found also in otherchlorophytes. These combinatorial iron scavenging and sparing strategies make Dunaliellaunique among photosynthetic organisms

iron homeostasis, phytoplankton, Iron starvation i↗
DOE OSTI2020

Structural and Proteomic Studies of the Aureococcus anophagefferens Virus Demonstrate a Global Distribution of Virus-Encoded Carbohydrate Processing

Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizing data to the per-virion level using a 9.5-Å-resolution molecular reconstruction of the 1900-Å (AaV) particle that we generated with cryogenic electron microscopy. This packaged proteome was used to determine similarities and differences between members of different giant virus families. We noted that proteins involved in sugar degradation and binding (e.g., carbohydrate lyases) were unique to AaV among characterized giant viruses. To determine the extent to which this virally encoded metabolic capability was ecologically relevant, we examined the TARA Oceans dataset and identified genes and transcripts of viral origin. Our analyses demonstrated that putative giant virus carbohydrate lyases represented up to 17% of the marine pool for this function. In total, our observations suggest that the AaV particle has potential prepackaged metabolic capabilities and that these may be found in other giant viruses that are widespread and abundant in global oceans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis , as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

FHOD formin and SRF promote post-embryonic striated muscle growth through separate pathways in C. elegans

Highlights: • Reduced SRF or formin FHOD-1 activity similarly inhibit muscle growth in worms. • Formin does not promote worm SRF-dependent muscle gene transcription. • Proteasome-driven myosin proteolysis is increased in worm muscle lacking formin. Previous work with cultured cells has shown transcription of muscle genes by serum response factor (SRF) can be stimulated by actin polymerization driven by proteins of the formin family. However, it is not clear if endogenous formins similarly promote SRF-dependent transcription during muscle development in vivo. We tested whether formin activity promotes SRF-dependent transcription in striated muscle in the simple animal model, Caenorhabditis elegans. Our lab has shown FHOD-1 is the only formin that directly promotes sarcomere formation in the worm's striated muscle. We show here FHOD-1 and SRF homolog UNC-120 both support muscle growth and also muscle myosin II heavy chain A expression. However, while a hypomorphic unc-120 allele blunts expression of a set of striated muscle genes, these genes are largely upregulated or unchanged by absence of FHOD-1. Instead, pharmacological inhibition of the proteasome restores myosin protein levels in worms lacking FHOD-1, suggesting elevated proteolysis accounts for their myosin deficit. Interestingly, proteasome inhibition does not restore normal muscle growth to fhod-1(Δ) mutants, suggesting formin contributes to muscle growth by some alternative mechanism. Overall, we find SRF does not depend on formin to promote muscle gene transcription in a simple in vivo system.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Abstract Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the green alga Chlamydomonas reinhardtii, Cu import is dependent on a transcription factor, Copper Response Regulator 1 (CRR1), responsible for activating genes in Cu-deficient cells. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulates the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high-affinity Cu(I) uptake. On the other hand, the overaccumulation of Cu(I) (20 times the quota) in zinc (Zn) deficiency depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and consistent with the lower substrate affinity of CTR2. One sentence summary Regulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

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

Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rβ1 as a receptor signaling subunit. In this paper, we present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rβ1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rβ2/IL-12Rβ1 and IL-23 receptor (IL-23R) complexes, which reveal “non-canonical” topologies where IL-12Rβ1 directly engages the common p40 subunit. We targeted the shared IL-12Rβ1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Rewiring the specificity of extracytoplasmic function sigma factors

Significance Bacterial phenotypes require the concerted expression of multiple genes, usually coordinated by a transcriptional regulator. Although the functions of many genes in sequenced bacterial genomes can be inferred, the regulatory networks that coordinate their expression are only known in a few model systems. Using a bioinformatic and experimental approach, we solve the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major class of bacterial regulators. We develop and use a high-stringency pipeline to predict the genes regulated by 67% of ECF σs in >10,000 species, providing a comprehensive look at the role of a broadly distributed family of gene regulatory proteins. This conceptual and computational framework is potentially applicable to other bacterial regulators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Osteocalcin binds to a GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A, a member of the Family C G-protein coupled receptors, regulates energy metabolism and sex hormone production and is activated by diverse ligands, including cations, L-amino acids, the osteocalcin (Ocn) peptide and the steroid hormone testosterone. We sought a structural framework for the ability of multiple distinct classes of ligands to active GPRC6A. We created a structural model of GPRC6A using Alphafold2. Using this model we explored a putative orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain of GPRC6A and two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7 transmembrane (7TM) domain. We provide evidence that Ocn peptides act as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this prediction, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the computationally predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations explain how dissimilar ligands activate GPRC6A and set the stage to develop novel molecules to activate and inhibit this previously poorly understood receptor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Review of Rattlesnake Venoms

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus, with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).

59 BASIC BIOLOGICAL SCIENCES↗