Fluoromethylketone-Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates.

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen.

Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains.

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumour development in a context-dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein.

Identification of Histone Peptide Binding Specificity and Small-Molecule Ligands for the TRIM33α and TRIM33β Bromodomains.

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMe) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches.

Structural basis of the zinc-induced cytoplasmic aggregation of the RNA-binding protein SFPQ.

SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm.

Structural analysis of phosphorylation-associated interactions of human MCC with Scribble PDZ domains.

Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbours a leucine-rich repeat domain and four PDZ domains that mediate most of Scribble's interactions with other proteins. It has become increasingly clear that post-translational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble.

A new crystal structure and small-angle X-ray scattering analysis of the homodimer of human SFPQ.

Splicing factor proline/glutamine-rich (SFPQ) is an essential RNA-binding protein that is implicated in many aspects of nuclear function. The structures of SFPQ and two paralogs, non-POU domain-containing octamer-binding protein and paraspeckle component 1, from the Drosophila behavior human splicing protein family have previously been characterized. The unusual arrangement of the four domains, two RNA-recognition motifs (RRMs), a conserved region termed the NonA/paraspeckle (NOPS) domain and a C-terminal coiled coil, in the intertwined dimer provides a potentially unique RNA-binding surface.

Structural and functional characterization of the membrane-permeabilizing activity of defensin NoD173 and protein engineering to enhance oncolysis.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics.

Crystal structure of the human Scribble PDZ1 domain bound to the PDZ-binding motif of APC.

Scribble (SCRIB) is an important adaptor protein that controls the establishment and maintenance of apico-basal cell polarity. To better understand how SCRIB controls cell polarity signalling via its PDZ domains, we investigated human SCRIB interactions with adenomatous polyposis coli (APC). We show that SCRIB PDZ1, PDZ2 and PDZ3 are the major interactors with the APC PDZ-binding motif (PBM), whereas SCRIB PDZ4 does not show detectable binding to APC.

A structural investigation of NRZ mediated apoptosis regulation in zebrafish.

Bcl-2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host-pathogen interactions and in disease. Dysregulation of Bcl-2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intra-family protein-protein interactions Bcl-2 family members regulate the release of cell death factors from mitochondria.

Human β-defensin 2 kills through phosphatidylinositol 4,5-bisphosphate-mediated membrane permeabilization.

Human defensins belong to a subfamily of the cationic antimicrobial peptides and act as a first line of defense against invading microbes. Their often broad-spectrum antimicrobial and antitumor activities make them attractive for therapeutic development; however, their precise molecular mechanism(s) of action remains to be defined. We show that human β-defensin 2 (HBD-2) permeabilizes cell membranes via a mechanism targeting the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP).

Grouper iridovirus GIV66 is a Bcl-2 protein that inhibits apoptosis by exclusively sequestering Bim.

Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode functional and structural homologs of Bcl-2 to counter premature host-cell apoptosis and ensure viral proliferation or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the Iridoviridae family and harbors GIV66, a putative Bcl-2-like protein and mitochondrially localized apoptosis inhibitor.

Guk-holder interacts with the Scribbled PDZ1 domain and regulates epithelial development with Scribbled and Discs Large.

Epithelial cell polarity is controlled by components of the Scribble polarity module, and its regulation is critical for tissue architecture and cell proliferation and migration. In , the adaptor protein Guk-holder (Gukh) binds to the Scribbled (Scrib) and Discs Large (Dlg) components of the Scribble polarity module and plays an important role in the formation of neuromuscular junctions. However, Gukh's role in epithelial tissue formation and the molecular basis for the Scrib-Gukh interaction remain to be defined.

The Bcl-2 Family in Host-Virus Interactions.

Members of the B cell lymphoma-2 (Bcl-2) family are pivotal arbiters of mitochondrially mediated apoptosis, a process of fundamental importance during tissue development, homeostasis, and disease. At the structural and mechanistic level, the mammalian members of the Bcl-2 family are increasingly well understood, with their interplay ultimately deciding the fate of a cell. Dysregulation of Bcl-2-mediated apoptosis underlies a plethora of diseases, and numerous viruses have acquired homologs of Bcl-2 to subvert host cell apoptosis and autophagy to prevent premature death of an infected cell.

Structural basis of apoptosis inhibition by the fowlpox virus protein FPV039.

Programmed cell death or apoptosis of infected host cells is an important defense mechanism in response to viral infections. This process is regulated by proapoptotic and prosurvival members of the B-cell lymphoma 2 (Bcl-2) protein family. To counter premature death of a virus-infected cell, poxviruses use a range of different molecular strategies including the mimicry of prosurvival Bcl-2 proteins.

Structural insight into an evolutionarily ancient programmed cell death regulator - the crystal structure of marine sponge BHP2 bound to LB-Bak-2.

Sponges of the porifera family harbor some of the evolutionary most ancient orthologs of the B-cell lymphoma-2 (Bcl-2) family, a protein family critical to regulation of apoptosis. The genome of the sponge Geodia cydonium contains the putative pro-survival Bcl-2 homolog BHP2, which protects sponge tissue as well as mammalian Hek-293 and NIH-3T3 cells against diverse apoptotic stimuli. The Lake Baikal demosponge Lubomirskia baicalensis has been shown to encode both putative pro-survival Bcl-2 (LB-Bcl-2) and pro-apoptotic Bcl-2 members (LB-Bak-2), which have been implied in axis formation (branches) in L.

Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis.

Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the family, and harbors A179L, a putative Bcl-2 like protein.

The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically Unstructured Region That Is Not Involved in Apoptosis Regulation.

Subversion of host cell apoptotic responses is a prominent feature of viral immune evasion strategies to prevent premature clearance of infected cells. Numerous poxviruses encode structural and functional homologs of the Bcl-2 family of proteins, and vaccinia virus harbors antiapoptotic F1L that potently inhibits the mitochondrial apoptotic checkpoint. Recently F1L has been assigned a caspase-9 inhibitory function attributed to an N-terminal α helical region of F1L spanning residues 1-15 (1) preceding the domain-swapped Bcl-2-like domains.

Structural basis of Deerpox virus-mediated inhibition of apoptosis.

Apoptosis is a key innate defence mechanism to eliminate virally infected cells. To counteract premature host-cell apoptosis, poxviruses have evolved numerous molecular strategies, including the use of Bcl-2 proteins, to ensure their own survival. Here, it is reported that the Deerpox virus inhibitor of apoptosis, DPV022, only engages a highly restricted set of death-inducing Bcl-2 proteins, including Bim, Bax and Bak, with modest affinities.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent.

Crystallization reports are the backbone of Acta Cryst. F, but do they have any spine?

Crystallization of macromolecules is famously difficult. By knowing what has worked for others, researchers can ease the process, both in the case where the protein has already been crystallized and in the situation where more general guidelines are needed. The 264 crystallization communications published in Acta Crystallographica Section F in 2012 have been reviewed, and from this analysis some information about trends in crystallization has been gleaned.

Crystallization and preliminary X-ray characterization of Epstein-Barr virus BHRF1 in complex with a benzoylurea peptidomimetic.

BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein-Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains.

Structural and mutational analyses of Deinococcus radiodurans UvrA2 provide insight into DNA binding and damage recognition by UvrAs.

UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms.