The basis for limited specificity and MHC restriction in a T cell receptor interface

αβ Tcell receptors (TCRs) recognize peptides presented by major histocompatibility complex (MHC) proteins using multiple complementarity-determining region (CDR) loops. TCRs display an array of poorly understood recognition properties, including specificity, crossreactivity and MHC restriction. Here we report a comprehensive thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A*0201, uncovering the physical basis for the receptor’s recognition properties. Broadly, our findings are in conflict with widely held generalities regarding TCR recognition, such as the relative contributions of central and peripheral peptide residues and the roles of the hypervariable and germline CDR loops in engaging peptide and MHC. Instead, we find that the recognition properties of the receptor emerge from the need to engage the composite peptide/MHC surface, with the receptor utilizing its CDR loops in a cooperative fashion such that specificity, crossreactivity and MHC restriction are inextricably linked

Structural diversity in the type IV pili of multidrug-resistant Acinetobacter

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating thatA. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter. Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCE: Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world\u27s population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus\u27s high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus

Structural and Evolutionary Analyses Show Unique Stabilization Strategies in the Type IV Pili of Clostridium difficile

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion and horizontal gene transfer. However, many Gram-positive species, including C. difficile, also produce Type IV pili. Here, we identify the major subunit of the Type IV pili of C. difficile, PilA1, and describe multiple three-dimensional structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative Type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the Type IVb pilins from Gram-negative bacteria that suggest that the Type IV pili of C. difficile are involved in microcolony formation

The Peculiar Nebula Simeis 57: I. Ionized Gas and Dust Extinction

We present high resolution radio continuum maps of the Galactic nebula Simeis 57 (= HS 191 = G 80.3+4.7) made with the WSRT and the DRAObservatory at frequencies of 609, 1412 and 1420 MHz. At optical and at radio wavelengths, the nebula has a peculiar ``S'' shape, crossed by long, thin and straight filaments. The radio maps, combined with other maps from existing databases, show essentially all radio emission from the peculiar and complex nebula to be thermal and optically thin. Although neither the distance nor the source of excitation of Simeis 57 are known, the nebula can only have a moderate electron density of typically n(e) = 100 cm-3. Its mass is also low, not exceeding some tens of solar masses. Peak emission measures are 5000 pc cm-6.Obscuring dust is closely associated with the nebula, but seems to occur mostly in front of it. Extinctions vary from A(V) = 1.0 mag to A(V) = 2.8 mag with a mean of about 2 mag. The extinction and the far-infrared emission at 100 microns are well-correlated.Comment: Accepted for Publication in Astronomy & Astrophysic

Structure of \u3ci\u3eClostridium difficile\u3c/i\u3e PilJ Exhibits Unprecedented Divergence from Known Type IV Pilins

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Grampositive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface

The NEU1-selective sialidase inhibitor, C9- butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-Nacetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 \u3e 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9- BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo

The Cygnus X region XXII. A probable HAeBe star with a giant bipolar outflow in DR 16

From medium-resolution radio images, DR 16 was suspected to be a large cometary nebula. To test this suggestion we obtained a higher resolution (15\arcsec) VLA continuum map. We also analyzed data from the Canadian Galactic Plane Survey in continuum, H I line, and IR. These data were supplemented by published near-infrared (J, H, K) stellar photometric results and MSX 8.28 micrometer data. We suggest that DR 16 is the diffuse H II region of an ongoing star formation site at a distance of about 3 kpc. The complicated radio picture arises from the superposition of diffuse H II with the remains of a giant bipolar outflow. The outflow was generated by a probable Herbig AeBe star, and the lobes are the remnants of its working surfaces. Additional ring-like features are discussed. DR 16 is part of a larger volume of space in the local spiral arm where star formation is an ongoing process.Comment: 15 pages, 14 figures, accepted by A&

First images of 6.7-GHz methanol masers in DR21(OH) and DR21(OH)N

The first images of 6.7-GHz methanol masers in the massive star-forming regions DR21(OH) and DR21(OH)N are presented. By measuring the shapes, radial velocities and polarization properties of these masers it is possible to map out the structure, kinematics and magnetic fields in the molecular gas that surrounds newly-formed massive stars. The intrinsic angular resolution of the observations was 43 mas (~100 AU at the distance of DR21), but structures far smaller than this were revealed by employing a non-standard mapping technique. This technique was used in an attempt to identify the physical structure (e.g. disc, outflow, shock) associated with the methanol masers. Two distinct star-forming centres were identified. In DR21(OH) the masers had a linear morphology, and the individual maser spots each displayed an internal velocity gradient in the same direction as the large-scale structure. They were detected at the same position as the OH 1.7-GHz ground-state masers, close to the centre of an outflow traced by CO and class I methanol masers. The shape and velocity gradients of the masers suggests that they probably delineate a shock. In DR21(OH)N the methanol masers trace an arc with a double-peaked profile and a complex velocity gradient. This velocity gradient closely resembles that of a Keplerian disc. The masers in the arc are 4.5% linearly polarized, with a polarization angle that indicates that the magnetic field direction is roughly perpendicular to the large-scale magnetic field in the region (indicated by lower angular resolution measurements of the CO and dust polarization). The suitability of channel-by-channel centroid mapping is discussed as an improved and viable means to maximise the information gained from the data.Comment: Accepted by MNRA

Generalized eigenfunctions and spectral theory for strongly local Dirichlet forms

We present an introduction to the framework of strongly local Dirichlet forms and discuss connections between the existence of certain generalized eigenfunctions and spectral properties within this framework. The range of applications is illustrated by a list of examples