Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase.

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

Ion channel chameleons: Switching ion selectivity by alternative splicing.

Voltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and an Na+-permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity.

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.

Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Graphene-Based Thermopile for Thermal Imaging Applications.

In this work, we leverage graphene's unique tunable Seebeck coefficient for the demonstration of a graphene-based thermal imaging system. By integrating graphene based photothermo-electric detectors with micromachined silicon nitride membranes, we are able to achieve room temperature responsivities on the order of ~7-9 V/W (at λ = 10.6 µm), with a time constant of ~23 ms. The large responsivities, due to the combination of thermal isolation and broadband infrared absorption from the underlying SiN membrane, have enabled detection as well as stand-off imaging of an incoherent blackbody target (300-500 K). By comparing the fundamental achievable performance of these graphene-based thermopiles with standard thermocouple materials, we extrapolate that graphene's high carrier mobility can enable improved performances with respect to two main figures of merit for infrared detectors: detectivity (>8 × 10(8) cm Hz(1/2) W(-1)) and noise equivalent temperature difference (<100 mK). Furthermore, even average graphene carrier mobility (<1000 cm(2) V(-1) s(-1)) is still sufficient to detect the emitted thermal radiation from a human target.

Photoresponse of an electrically tunable ambipolar graphene infrared thermocouple.

We explore the photoresponse of an ambipolar graphene infrared thermocouple at photon energies close to or below monolayer graphene's optical phonon energy and electrostatically accessible Fermi energy levels. The ambipolar graphene infrared thermocouple consists of monolayer graphene supported by an infrared absorbing material, controlled by two independent electrostatic gates embedded below the absorber. Using a scanning infrared laser microscope, we characterize these devices as a function of carrier type and carrier density difference controlled at the junction between the two electrostatic gates. On the basis of these measurements, conducted at both mid- and near-infrared wavelengths, the primary detection mechanism can be modeled as a thermoelectric response. By studying the effect of different infrared absorbers, we determine that the optical absorption and thermal conduction of the substrate play the dominant role in the measured photoresponse of our devices. These experiments indicate a path toward hybrid graphene thermal detectors for sensing applications such as thermography and chemical spectroscopy.

Dielectric screening of excitons and trions in single-layer MoS2.

Photoluminescence (PL) properties of single-layer MoS2 are indicated to have strong correlations with the surrounding dielectric environment. Blue shifts of up to 40 meV of exciton or trion PL peaks were observed as a function of the dielectric constant of the environment. These results can be explained by the dielectric screening effect of the Coulomb potential; based on this, a scaling relationship was developed with the extracted electronic band gap and exciton and trion binding energies in good agreement with theoretical estimations. It was also observed that the trion/exciton intensity ratio can be tuned by at least 1 order of magnitude with different dielectric environments. Our findings are helpful to better understand the tightly bound exciton properties in strongly quantum-confined systems and provide a simple approach to the selective and separate generation of excitons or trions with potential applications in excitonic interconnects and valleytronics.

Iron (III) chloride doping of CVD graphene.

Chemical doping has been shown as an effective method of reducing the sheet resistance of graphene. We present the results of our investigations into doping large area chemical vapor deposition graphene using Iron (III) Chloride (FeCl(3)). It is shown that evaporating FeCl(3) can increase the carrier concentration of monolayer graphene to greater than 10(14) cm(-2) and achieve resistances as low as 72 Ω sq(-1). We also evaluate other important properties of the doped graphene such as surface cleanliness, air stability, and solvent stability. Furthermore, we compare FeCl(3) to three other common dopants: Gold (III) Chloride (AuCl(3)), Nitric Acid (HNO(3)), and TFSA ((CF(3)SO(2))(2)NH). We show that compared to these dopants, FeCl(3) can not only achieve better sheet resistance but also has other key advantages including better solvent stability.