Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells.

Identification of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C.

Addressing Needs of Long-Term Care Facility Residents During Acute Hospitalization.

Patients who reside in long-term care facilities for extended periods of time offer many health care challenges. Recognition of early signs of illness, care transitions, and underutilization of community resources are common concerns.

Inhibition of oncogenic Wnt signaling through direct targeting of β-catenin.

Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. Owing to the persistent dependence of these tumors on Wnt signaling for growth and survival, inhibition of this pathway is considered an attractive mechanism-based therapeutic approach. Oncogenic activation of Wnt signaling can ensue from a variety of distinct aberrations in the signaling pathway, but most share the common feature of causing increased cellular levels of β-catenin by interfering with its constitutive degradation.

Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1.

A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed.

Toward Visualization for Games: Theory, Design Space, and Patterns.

Electronic games are starting to incorporate in-game telemetry that collects data about player, team, and community performance on a massive scale, and as data begins to accumulate, so does the demand for effectively analyzing this data. In this paper, we use examples from both old and new games of different genres to explore the theory and design space of visualization for games. Drawing on these examples, we define a design space for this novel research topic and use it to formulate design patterns for how to best apply visualization technology to games.

Regenerative protein thymosin beta-4 is a novel regulator of purinergic signaling.

By an unknown mechanism, β-thymosins are extracellular modulators of angiogenesis, inflammation, wound healing, and development. We were interested in identifying β-thymosin interactors and determining their importance in β-thymosins signaling in human vein endothelial cells (HUVECs). We performed pulldown experiments with biotinylated thymosin β-4 (Tβ4) in comparison to neutravidin beads alone and used mass spectrometric analysis to identify differentially interacting proteins.

Structure of Escherichia coli AlkA in complex with undamaged DNA.

Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA.

Synthesis and structure of duplex DNA containing the genotoxic nucleobase lesion N7-methylguanine.

The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2'-deoxyguanosine (m7dG). M7dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans.

Structure of the E. coli DNA glycosylase AlkA bound to the ends of duplex DNA: a system for the structure determination of lesion-containing DNA.

The constant attack on DNA by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. Visualizing the effects of these lesions on the structure of duplex DNA is key to understanding their biologic consequences. The most definitive method of obtaining such structures, X-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of DNA.

Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding.

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.

X-ray structures of the N- and C-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation.

Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways.

Crystal structures of the vaccinia virus polyadenylate polymerase heterodimer: insights into ATP selectivity and processivity.

Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition.

Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1.

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.

Multipurpose MRG domain involved in cell senescence and proliferation exhibits structural homology to a DNA-interacting domain.

The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the approximately 20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins.

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2.

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins.

Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics.

The dispositions of 39 alpha helices of greater than 2.5 turns and four beta sheets in the major capsid protein (VP5, 149 kDa) of herpes simplex virus type 1 were identified by computational and visualization analysis from the 8.5A electron cryomicroscopy structure of the whole capsid.

Structure of the herpesvirus major capsid protein.

Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus.