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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 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Structural basis of unidirectional export of lipopolysaccharide to the cell surface

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown. In this paper we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A coevolution-guided model for the rotor of the bacterial flagellar motor

The Salmonella typhimurium trans-membrane FliF MS ring templates assembly of the rotary bacterial fagellar motor, which also contains a cytoplasmic C-ring. A full-frame fusion of FliF with the rotor protein FliG assembles rings in non-motile expression hosts. 3D electron microscopy reconstructions of these FliFFliG rings show three high electron-density sub-volumes. 3D-classifcation revealed heterogeneity of the assigned cytoplasmic volume consistent with FliG lability. We used residue coevolution to construct homodimer building blocks for ring assembly, with X-ray crystal structures from other species and injectisome analogs. The coevolution signal validates folds and, importantly, indicates strong homodimer contacts for three ring building motifs (RBMs), initially identified in injectisome structures. It also indicates that the cofolded domains of the FliG N-terminal domain (FliG_N) with embedded α-helical FliF carboxy-terminal tail homo-oligomerize. The FliG middle and C-terminal domains (FliG_MC) have a weak signal for homo-dimerization but have coevolved to conserve their stacking contact. The homodimers and their ring models ft well into the 3D reconstruction. We hypothesize that a stable FliF periplasmic hub provides a platform for FliG ring self-assembly, but the FliG_MC ring has only limited stability without the C-ring. We also present a mechanical model for torque transmission in the FliFFliG ring.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Fusion and fission events regulate endosome maturation and viral escape

Endosomes are intracellular vesicles that mediate the communication of the cell with its extracellular environment. They are an essential part of the cell’s machinery regulating intracellular trafficking via the endocytic pathway. Many viruses, which in order to replicate require a host cell, attach themselves to the cellular membrane; an event which usually initiates uptake of a viral particle through the endocytic pathway. In this way viruses hijack endosomes for their journey towards intracellular sites of replication and avoid degradation without host detection by escaping the endosomal compartment. Recent experimental techniques have defined the role of endosomal maturation in the ability of enveloped viruses to release their genetic material into the cytoplasm. Endosome maturation depends on a family of small hydrolase enzymes (or GTPases) called Rab proteins, arranged on the cytoplasmic surface of its membrane. Here, we model endosomes as intracellular compartments described by two variables (its levels of active Rab5 and Rab7 proteins) and which can undergo coagulation (or fusion) and fragmentation (or fission). The key element in our approach is the “per-cell endosomal distribution” and its dynamical (Boltzmann) equation. The Boltzmann equation allows us to derive the dynamics of the total number of endosomes in a cell, as well as the mean and the standard deviation of its active Rab5 and Rab7 levels. We compare our mathematical results with experiments of Dengue viral escape from endosomes. The relationship between endosomal active Rab levels and pH suggests a mechanism that can account for the observed variability in viral escape times, which in turn regulate the viability of a viral intracellular infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment

Significance Mechanisms that determine the cellular volume allocated to organelles are largely unknown. We demonstrate that in the plant Arabidopsis thaliana , a small gene family that encodes proteins of unknown function contributes to a mechanism that establishes the proportion of cellular volume devoted to chloroplasts. We show that this mechanism resides outside of the chloroplast by demonstrating that the protein that makes the greatest contribution to this mechanism resides in the cytoplasm and nucleus and that the trafficking of this protein between the cytoplasm and nucleus may regulate this mechanism. A deeper understanding of this mechanism may lead to the rational manipulation of chloroplast compartment size, which may lead to more efficient photosynthesis and increased yields from important crop plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Cytoplasmic calcium (Ca 2+ ) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca 2+ -independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca 2+ activation and ion selectivity. A “Ca 2+ clasp” within the channel’s intracellular region acts as a sensor of cytoplasmic Ca 2+ . Alanine substitutions within a hydrophobic “neck” of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca 2+ . We conclude that the primary function of the neck is as a “gate” that controls chloride permeation in a Ca 2+ -dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel’s ion selectivity. We find that mutation of a cytosolic “aperture” of the pore does not perturb the Ca 2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Furthermore, unlike ion channels that have a single “selectivity filter,” in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as “phosphosites”) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

Science & Technology - Other Topics↗
DOE OSTI2018

Autonomous conformational regulation of β 3 integrin and the conformation-dependent property of HPA-1a alloantibodies

Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The β 3 integrins, including α IIb β 3 and α V β 3 , are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β 3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti–HPA-1a alloantibodies can distinguish the α IIb β 3 from α V β 3 and affect their functions with unknown mechanisms. Here we designed a single-chain β 3 subunit that mimics a separation of α/β heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β 3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β 3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β 3 integrin, we identified the conformation-dependent property of anti–HPA-1a alloantibodies, which enables them to differently recognize the β 3 in the bent state vs. the extended state and in the complex with α IIb vs. α V . Furthermore, this study provides deeper understandings of integrin conformational activation on the cell surface.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. In this work, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to β-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td) . The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable μ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III] 2- O 2 2– , Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular characterization of a fungal gasdermin-like protein

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 ( rcd-1 ), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa . rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2 , is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1 -dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1–1/rcd-1–2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 . This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ~90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

KRAS↗
DOE OSTI2015

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin. Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue

Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

Majumdar, Shreoshi↗
DOE OSTI2026

From photosynthetic electron flow to gene regulation: redox signal transduction in cyanobacteria

In cyanobacteria, the free-living ancestors of chloroplasts, photosynthesis simultaneously sustains growth and generates reactive oxygen species (ROS) that damage proteins, lipids, and DNA when light capture outpaces carbon fixation. Maintaining redox balance, therefore, requires cells to read photosynthetic electron flow as a signal that continuously tunes gene expression and protein activity. This review traces how these redox signals are transduced to transcription machinery through three routes: membrane-localized sensors, cytoplasmic redox sensors downstream of photosystem I, and ROS generated when electron sinks are saturated. Membrane-bound histidine kinases (two-component systems) relay the redox state of the plastoquinone pool to control photosystem remodeling, pigment biosynthesis, and circadian timing. Cytoplasmic one-component regulators, by contrast, sense redox directly through thiol-disulfide switches, glutathionylation, iron-sulfur clusters, and metal-catalyzed oxidation to control photosystem-cofactor, electron-carrier, and transition-metal homeostasis. Because many of these regulators persist in algal and plant chloroplasts, cyanobacteria illuminate principles of redox control across photosynthetic eukaryotes. Post-transcriptional and translational control further shapes redox-dependent gene expression programs through transcript stability, ribosome assembly, and translation initiation, extending redox regulation beyond transcription to every step of protein synthesis and even activity modulation. Finally, we connect redox regulation to photosynthetic physiology, stress resilience, and the rational engineering of cyanobacteria for sustainable bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Packaging “vegetable oils”: Insights into plant lipid droplet proteins

Abstract Plant neutral lipids, also known as “vegetable oils”, are synthesized within the endoplasmic reticulum (ER) membrane and packaged into subcellular compartments called lipid droplets (LDs) for stable storage in the cytoplasm. The biogenesis, modulation, and degradation of cytoplasmic LDs in plant cells are orchestrated by a variety of proteins localized to the ER, LDs, and peroxisomes. Recent studies of these LD-related proteins have greatly advanced our understanding of LDs not only as steady oil depots in seeds but also as dynamic cell organelles involved in numerous physiological processes in different tissues and developmental stages of plants. In the past 2 decades, technology advances in proteomics, transcriptomics, genome sequencing, cellular imaging and protein structural modeling have markedly expanded the inventory of LD-related proteins, provided unprecedented structural and functional insights into the protein machinery modulating LDs in plant cells, and shed new light on the functions of LDs in nonseed plant tissues as well as in unicellular algae. Here, we review critical advances in revealing new LD proteins in various plant tissues, point out structural and mechanistic insights into key proteins in LD biogenesis and dynamic modulation, and discuss future perspectives on bridging our knowledge gaps in plant LD biology.

Cai, Yingqi (ORCID:0000000203575809)↗
DOE OSTI2018

Engineering DNA nanotubes for resilience in an E. coli TXTL system

Abstract Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.

Klocke, Melissa A.↗
DOE OSTI2015

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

Elevated carbon dioxide (CO 2 ) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO 2 -induced stomatal movements by catalytic transmission of the CO 2 signal in guard cells. But, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO 2 -regulated stomatal movements remains unknown. We express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO 2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO 2 regulation of stomatal conductance. Mathematical modeling of cellular CO 2 catalysis suggests that the dynamics of the intracellular HCO 3 - concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Therefore, modeling supports the notion that the intracellular HCO 3 - concentration dynamics in guard cells are a key mechanism in mediating CO 2 -regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Defective Pollen Wall 2 ( DPW2 ) Encodes an Acyl Transferase Required for Rice Pollen Development

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using v-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:v-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗