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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 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

A revised partiality model and post-refinement algorithm for X-ray free-electron laser data

An updated partiality model and post-refinement algorithm for XFEL snapshot diffraction data is presented and confirmed by observing anomalous density for S atoms at an X-ray wavelength of 1.3 Å. Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the R{sub split} value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2015

Structural conservation of chemotaxis machinery across A rchaea and B acteria

Summary Chemotaxis allows cells to sense and respond to their environment. In B acteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two‐component regulatory system. These arrays take the form of highly stereotyped super‐lattices comprising hexagonally packed trimers‐of‐receptor‐dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic B acteria, and between membrane‐bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in A rchaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of E uryarchaeota. Unlike in B acteria, the chemotaxis system then evolved largely vertically in A rchaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane‐bound and cytoplasmic chemoreceptor arrays is conserved between B acteria and A rchaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles.

Briegel, Ariane↗
DOE OSTI2016

Spermatogonial stem cell organization in felid testis as revealed by Dolichos biflorus lectin

Summary Spermatogonial stem cells are being exploited in many species as a tool to recover fertility, but may also be used to manipulate the genetic pool. Whatever the purpose, these cells must be fully characterized and easily identifiable, and our goal was to improve this procedure in the domestic cat, used as an animal model for endangered felid species and for some human diseases/physiological processes. We have therefore screened several markers that might be used to distinguish and study the undifferentiated spermatogonia population in situ and in vitro via immunohistochemistry applied to tissue sections and whole mounts of the domestic cat seminiferous tubules. Our results show that, although they label the cytoplasm and nucleus of gonocytes and spermatogonia in pre‐pubertal animals, PGP 9.5 and FoxO1 cannot be considered markers of undifferentiated spermatogonia in adult animals, as almost all spermatogonia, namely type A and B, express these proteins. Nonetheless, the Dolichos biflorus agglutinin ( DBA lectin) was able to label the cell surface and cytoplasm of a small type A spermatogonial population in the adult animals. Analysis of the number and distribution of the DBA ‐labeled cells showed they were present in low number, which did not vary with epithelium seminiferous stage. Morphometric analysis revealed that DBA ‐labeled cells present tropism to a peculiar area of the seminiferous tubules, namely the area in direct contact with Leydig cells. Whole mounts of DBA ‐stained seminiferous tubules revealed the arrangement of DBA ‐stained cells in small clones up to eight cells. Noteworthy, the clonal cells presented variable staining intensity suggesting the existence of asymmetric distribution of O‐glycosylated proteins within each clone. Our results strongly suggest that the DBA lectin is a marker of undifferentiated spermatogonia in domestic cat, and illustrate the peculiar characteristics of spermatogonial stem cell development and organization in this species.

Escada‐Rebelo, S.↗
DOE OSTI2020

Auxin‐induced actin cytoskeleton rearrangements require AUX1

Summary The actin cytoskeleton is required for cell expansion and implicated in cellular responses to the phytohormone auxin. However, the mechanisms that coordinate auxin signaling, cytoskeletal remodeling and cell expansion are poorly understood. Previous studies examined long‐term actin cytoskeleton responses to auxin, but plants respond to auxin within minutes. Before this work, an extracellular auxin receptor – rather than the auxin transporter AUXIN RESISTANT 1 (AUX1) – was considered to precede auxin‐induced cytoskeleton reorganization. In order to correlate actin array organization and dynamics with degree of cell expansion, quantitative imaging tools established baseline actin organization and illuminated individual filament behaviors in root epidermal cells under control conditions and after indole‐3‐acetic acid (IAA) application. We evaluated aux1 mutant actin organization responses to IAA and the membrane‐permeable auxin 1‐naphthylacetic acid (NAA). Cell length predicted actin organization and dynamics in control roots; short‐term IAA treatments stimulated denser and more parallel, longitudinal arrays by inducing filament unbundling within minutes. Although AUX1 is necessary for full actin rearrangements in response to auxin, cytoplasmic auxin (i.e. NAA) stimulated a lesser response. Actin filaments became more ‘organized’ after IAA stopped elongation, refuting the hypothesis that ‘more organized’ actin arrays universally correlate with rapid growth. Short‐term actin cytoskeleton response to auxin requires AUX1 and/or cytoplasmic auxin.

AUX1↗
DOE OSTI2024

Cortical cell size regulates root metabolic cost

SUMMARY It has been hypothesized that vacuolar occupancy in mature root cortical parenchyma cells regulates root metabolic cost and thereby plant fitness under conditions of drought, suboptimal nutrient availability, and increased soil mechanical impedance. However, the mechanistic role of vacuoles in reducing root metabolic cost was unproven. Here we provide evidence to support this hypothesis. We first show that root cortical cell size is determined by both cortical cell diameter and cell length. Significant genotypic variation for both cortical cell diameter (~1.1‐ to 1.5‐fold) and cortical cell length (~ 1.3‐ to 7‐fold) was observed in maize and wheat. GWAS and QTL analyses indicate cortical cell diameter and length are heritable and under independent genetic control. We identify candidate genes for both phenes. Empirical results from isophenic lines contrasting for cortical cell diameter and length show that increased cell size, due to either diameter or length, is associated with reduced root respiration, nitrogen content, and phosphorus content.RootSlice, a functional‐structural model of root anatomy, predicts that an increased vacuolar: cytoplasmic ratio per unit cortical volume causes reduced root respiration and nutrient content. Ultrastructural imaging of cortical parenchyma cells with varying cortical diameter and cortical cell length confirms thein silicopredictions and shows that an increase in cell size is correlated with increased vacuolar volume and reduced cytoplasmic volume. Vacuolar occupancy and its relationship with cell size merits further investigation as a phene for improving crop adaptation to edaphic stress.

Plant Sciences↗
DOE OSTI2016

Sci-Thur AM: YIS – 06: A Monte Carlo study of macro- and microscopic dose descriptors and the microdosimetric spread using detailed cellular models

Purpose: To develop Monte Carlo models of cell clusters to investigate the relationships between macro- and microscopic dose descriptors, quantify the microdosimetric spread in energy deposition for subcellular targets, and determine how these results depend on the computational model. Methods: Microscopic tissue structure is modelled as clusters of 13 to 150 cells, with cell (nuclear) radii between 5 and 10 microns (2 and 9 microns). Energy imparted per unit mass (specific energy or dose) is scored in the nucleus (D{sub nuc}) and cytoplasm (D{sub cyt}) for incident photon energies from 20 to 370 keV. Dose-to-water (D{sub w,m}) and dose-to-medium (D{sub m,m}) are compared to D{sub nuc} and D{sub cyt}. Single cells and single nuclear cavities are also simulated. Results: D{sub nuc} and D{sub cyt} are sensitive to the surrounding environment with deviations of up to 13% for a single nucleus/cell compared with a multicellular cluster. These dose descriptors vary with cell and nucleus size by up to 10%. D{sub nuc} and D{sub cyt} differ from D{sub w,m} and D{sub m,m} by up to 32%. The microdosimetric spread is sensitive to whether cells are arranged randomly or in a hexagonal lattice, and whether subcellular compartment sizes are sampled from a normal distribution or are constant throughout the cluster. Conclusions: D{sub nuc} and D{sub cyt} are sensitive to cell morphology, elemental composition and the presence of surrounding cells. The microdosimetric spread was investigated using realistic elemental compositions for the nucleus and cytoplasm, and depends strongly on subcellular compartment size, source energy and dose.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source

ABSTRACT Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures (“py”’) demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c - and b -type cytochromes was observed in “py,” pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells’ photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(−), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet’s history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sensor Domain of Histidine Kinase VxrA of Vibrio cholerae : Hairpin-Swapped Dimer and Its Conformational Change

VxrA and VxrB are cognate histidine kinase (HK)-response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae, a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the type VI secretion system, biofilm formation, and cell wall homeostasis in V. cholerae, providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. Here, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual β hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK. IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the type six secretion system, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structures of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown; however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Structure-Function Analysis of Vaccinia Virus H7 Protein Reveals a Novel Phosphoinositide Binding Fold Essential for Poxvirus Replication

Phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide binding domains. Recently, a group of poxvirus proteins, termed viral membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven α-helices and a highly curved three-stranded antiparallel β-sheet. We identified a phosphoinositide binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for viral replication and for binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggest that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bacteria export alarmone synthetases that produce (p)ppApp and (p)ppGpp

Guanosine penta- and tetraphosphate [(p)ppGpp] and their adenosine analogs [(p)ppApp] are bacterial second messengers known as alarmones. Members of the RelA-SpoT homolog (RSH) family synthesize (p)ppGpp to mediate the stringent response during nutrient starvation, whereas (p)ppApp synthetases have been identified as bactericidal toxins in diverse contexts including type VI secretion systems, toxin-antitoxin modules, and phages. Although alarmone synthesis has traditionally been viewed as a cytoplasmic process, early studies in Streptomyces suggested the existence of secreted alarmone synthetases. Here, we identify SaEAS, an exported alarmone synthetase (EAS) from Streptomyces albidoflavus, as the long-mysterious source of extracellular alarmone synthetase activity in Streptomyces. SaEAS produces both (p)ppGpp and (p)ppApp at rates exceeding 100,000 molecules per minute and has kinetic properties adapted to low substrate environments. A broader bioinformatic survey reveals ~600 EASs linked to a range of specialized bacterial secretion systems. Characterization of two additional EASs, VpEAS from Vibrio parahaemolyticus and AaEAS from Amycolatopsis azurea, shows that both produce (p)ppGpp exclusively and inhibit bacterial growth when localized to the cytoplasm. These findings challenge the longstanding view of (p)ppGpp as strictly pro-survival and unveil a diverse family of secreted RSH enzymes with potential roles in interbacterial antagonism and environmental signaling.

Ahmad, Shehryar↗
DOE OSTI2014

Reconstruction and modeling protein translocation and compartmentalization in Escherichia coli at the genome-scale

Background: Membranes play a crucial role in cellular functions. Membranes provide a physical barrier, control the trafficking of substances entering and leaving the cell, and are a major determinant of cellular ultra-structure. In addition, components embedded within the membrane participate in cell signaling, energy transduction, and other critical cellular functions. All these processes must share the limited space in the membrane; thus it represents a notable constraint on cellular functions. Membrane- and location-based processes have not yet been reconstructed and explicitly integrated into genome-scale models. Results: The recent genome-scale model of metabolism and protein expression in Escherichia coli (called a ME-model) computes the complete composition of the proteome required to perform whole cell functions. Here we expand the ME-model to include (1) a reconstruction of protein translocation pathways, (2) assignment of all cellular proteins to one of four compartments (cytoplasm, inner membrane, periplasm, and outer membrane) and a translocation pathway, (3) experimentally determined translocase catalytic and porin diffusion rates, and (4) a novel membrane constraint that reflects cell morphology. Comparison of computations performed with this expanded ME-model, named iJL1678-ME, against available experimental data reveals that the model accurately describes translocation pathway expression and the functional proteome by compartmentalized mass. Conclusion: iJL1678-ME enables the computation of cellular phenotypes through an integrated computation of proteome composition, abundance, and activity in four cellular compartments (cytoplasm, periplasm, inner and outer membrane). Reconstruction and validation of the model has demonstrated that the iJL1678-ME is capable of capturing the functional content of membranes, cellular compartment-specific composition, and that it can be utilized to examine the effect of perturbing an expanded set of network components. iJL1678-ME takes a notable step towards the inclusion of cellular ultra-structure in genome-scale models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2013

Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

Introduction: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, biomarker discovery from body fluids is often hampered by the high abundance of many proteins unrelated to disease. An attractive alternative biomarker discovery approach is the isolation of small vesicles (exosomes, ~100 nm). They contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific marker discovery. Profiling prostate cancer-derived exosomes could reveal new markers for this malignancy. Materials and Methods: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. Proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode, followed by the Accurate Mass and Time (AMT) tag approach. Exosomal proteins were validated by Western blotting. A Tissue Micro Array, containing 481 different PCa samples (radical prostatectomy), was used to correlate candidate markers with several clinical-pathological parameters such as PSA, Gleason score, biochemical recurrence, and (PCa-related) death. Results: Proteomic characterization resulted in the identification of 263 proteins by at least 2 peptides. Specifically analysis of exosomes from PNT2C2, RWPE-1, PC346C, and VCaP identified 248, 233, 169, and 216 proteins, respectively. Statistical analyses revealed 52 proteins differently expressed between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. The Tissue Micro 4 Array showed strong correlation of higher Gleason scores and local recurrence with increased cytoplasmic XPO1 (P<0.001). Conclusions: Differentially abundant proteins of cell line-derived exosomes make a clear subdivision between benign and malignant origin. Validation showed a preferential abundance of PDCD6IP, FASN and XPO1. Cytoplasmic XPO1 is the most promising candidate biomarker.

biomarkers↗
DOE OSTI2014

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na + and K + ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na + and K + . These structural rearrangements facilitate entry of K + ions into the selectivity filter and permeation through the channel, and rejection of Na + ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K + . Estimates of the K + /Na + selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na + ions, the ‘‘punch through’’ relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.

36 MATERIALS SCIENCE↗