Structural Basis for the Restoration of TCR Recognition of an MHC Allelic Variant by Peptide Secondary Anchor Substitution

Major histocompatibility complex (MHC) class I variants H-2Kb and H-2Kbm8 differ primarily in the B pocket of the peptide-binding groove, which serves to sequester the P2 secondary anchor residue. This polymorphism determines resistance to lethal herpes simplex virus (HSV-1) infection by modulating T cell responses to the immunodominant glycoprotein B498-505 epitope, HSV8. We studied the molecular basis of these effects and confirmed that T cell receptors raised against Kb–HSV8 cannot recognize H-2Kbm8–HSV8. However, substitution of SerP2 to GluP2 (peptide H2E) reversed T cell receptor (TCR) recognition; H-2Kbm8–H2E was recognized whereas H-2Kb–H2E was not. Insight into the structural basis of this discrimination was obtained by determining the crystal structures of all four MHC class I molecules in complex with bound peptide (pMHCs). Surprisingly, we find no concerted pMHC surface differences that can explain the differential TCR recognition. However, a correlation is apparent between the recognition data and the underlying peptide-binding groove chemistry of the B pocket, revealing that secondary anchor residues can profoundly affect TCR engagement through mechanisms distinct from the alteration of the resting state conformation of the pMHC surface

Jet-Induced Emission-Line Nebulosity and Star Formation in the High-Redshift Radio Galaxy 4C41.17

The high redshift radio galaxy 4C41.17 consists of a powerful radio source in which previous work has shown that there is strong evidence for jet-induced star formation along the radio axis. We argue that nuclear photoionization is not responsible for the excitation of the emission line clouds and we construct a jet-cloud interaction model to explain the major features revealed by the data. The interaction of a high-powered jet with a dense cloud in the halo of 4C41.17 produces shock-excited emission-line nebulosity through ~1000 km/s shocks and induces star formation. The CIII to CIV line ratio and the CIV luminosity emanating from the shock, imply that the pre-shock density in the line-emitting cloud is high enough (~1-10 cm^-3) that shock initiated star formation could proceed on a timescale of order a few x 10^6 yrs, well within the estimated dynamical age of the radio source. Broad (FWHM ~ 100 - 1400 km/s) emission lines are attributed to the disturbance of the gas cloud by a partial bow--shock and narrow emission lines (FWHM ~ 500 - 650 km/s) (in particular CIV) arise in precursor emission in relatively low metallicity gas. The implied baryonic mass ~ 8 \times 10^{10} solar masses of the cloud is high and implies that Milky Way size condensations existed in the environments of forming radio galaxies at a redshift of 3.8. Our interpretation of the data provides a physical basis for the alignment of the radio, emission-line and UV continuum images in some of the highest redshift radio galaxies and the analysis presented here may form a basis for the calculation of densities and cloud masses in other high redshift radio galaxies.Comment: 18 pages, 5 figures; uses astrobib.sty and aaspp4.sty. Better versions of figures available via anonymous from ftp://mso.anu.edu.au:pub/pub/geoff/4C41.1

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme

Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development

In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers

Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development

The crystal structure of human UDP-glucuronosyltransferase 2B7 C-terminal end is the first mammalian UGT target to be revealed: the significance for human UGTs from both the 1A and 2B families

Human UDP-glucuronosyltransferases (EC 2.4.1.17) (UGTs) are major phase II metabolism enzymes that detoxify a multitude of endo- and xenobiotics through the covalent addition of a glucuronic acid moiety. UGTs are promiscuous enzymes that regulate the levels of numerous important endobiotics in a range of tissues, and inactivate most therapeutic compounds in concert with phase I enzymes. In spite of the importance of these enzymes, we have only a limited understanding of the molecular mechanisms governing their substrate specificity and catalytic activity. Until recently, no three-dimensional structural information was available for any mammalian UGT. The 1.8-Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT2B7 (2B7CT) is the only structure of a mammalian UGT target determined to date. In this review, we summarize what has been learned about human UGT function from the analysis of this and other related glycosyltransferase (GT) crystal structures

Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 1-2 Å, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point > 140 °C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 Å)

Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 μM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα RMSD = 1.4 Å)

Giant Lya nebulae associated with high redshift radio galaxies

We report deep Keck narrow-band Lya images of the luminous z > 3 radio galaxies 4C 41.17, 4C 60.07, and B2 0902+34. The images show giant, 100-200 kpc scale emission line nebulae, centered on these galaxies, which exhibit a wealth of morphological structure, including extended low surface brightness emission in the outer regions, radially directed filaments, cone-shaped structures and (indirect) evidence for extended Lya absorption. We discuss these features within a general scenario where the nebular gas cools gravitationally in large Cold Dark Matter (CDM) halos, forming stars and multiple stellar systems. Merging of these ``building'' blocks triggers large scale starbursts, forming the stellar bulges of massive radio galaxy hosts, and feeds super-massive black holes which produce the powerful radio jets and lobes. The radio sources, starburst superwinds and AGN radiation then disrupt the accretion process limiting galaxy and black hole growth, and imprint the observed filamentary and cone-shaped structures of the Lya nebulae.Comment: 36 Pages, including 8 Postscript figures. Accepted for publication in the Astrophysical Journa

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MerTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MerTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket

Crystal Structure of the Cofactor-Binding Domain of the Human Phase II Drug-Metabolism Enzyme UDP-Glucuronosyltransferase 2B7

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in the processing of a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8 Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designed UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicate that human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting that human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer