Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69.

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex.

Crystal structure of the Ube2K/E2-25K and K48-linked di-ubiquitin complex provides structural insight into the mechanism of K48-specific ubiquitin chain synthesis.

Ubiquitin-conjugating enzymes (E2) form thioester bonds with ubiquitin (Ub), which are subsequently transferred to target proteins for cellular progress. Ube2K/E2-25K (a class II E2 enzyme) contains a C-terminal ubiquitin-associated (UBA) domain that has been suggested to control ubiquitin recognition, dimerization, or poly-ubiquitin chain formation. Ube2K is a special E2 because it synthesizes K48-linked poly-ubiquitin chains without E3 ubiquitin ligase.

Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanI) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer.

Structural implications of Ca-dependent actin-bundling function of human EFhd2/Swiprosin-1.

EFhd2/Swiprosin-1 is a cytoskeletal Ca-binding protein implicated in Ca-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region.

Structural insights into the interaction of human p97 N-terminal domain and SHP motif in Derlin-1 rhomboid pseudoprotease.

The interaction of the rhomboid pseudoprotease Derlin-1 and p97 is crucial for the retrotranslocation of polyubiquitinated substrates in the endoplasmic reticulum-associated degradation pathway. We report a 2.25 Å resolution structure of the p97 N-terminal domain (p97N) in complex with the Derlin-1 SHP motif.

Structural insights into the interaction of p97 N-terminus domain and VBM in rhomboid protease, RHBDL4.

RHBDL4 is an active rhomboid that specifically recognizes and cleaves atypical, positively charged transmembrane endoplasmic reticulum-associated degradation (ERAD) substrates. Interaction of valosin-containing protein (p97/VCP) and RHBDL4 is crucial to retrotranslocate polyubiquitinated substrates for ERAD pathway. Here, we report the first complex structure of VCP-binding motif (VBM) with p97 N-terminal domain (p97N) at 1.

Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis.

Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear.

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.

Structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors. Corrigendum.

A correction is made to the article by Lee et al. [(2014) Acta Cryst. D70, 1357-1365].

Structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors.

Sialidase catalyzes the removal of a terminal sialic acid from glycoconjugates and plays a pivotal role in nutrition, cellular interactions and pathogenesis mediating various infectious diseases including cholera, influenza and sepsis. An array of antiviral sialidase agents have been developed and are commercially available, such as zanamivir and oseltamivir for treating influenza. However, the development of bacterial sialidase inhibitors has been much less successful.

Crystal structure of the N-terminal domain of MinC dimerized via domain swapping.

Proper cell division at the mid-site of gram-negative bacteria reflects critical regulation by the min system (MinC, MinD and MinE) of the cytokinetic Z ring, which is a polymer composed of FtsZ subunits. MinC and MinD act together to inhibit aberrantly positioned Z-ring formation. MinC consists of two domains: an N-terminal domain (MinCNTD), which interacts with FtsZ and inhibits FtsZ polymerization, and a C-terminal domain (MinCCTD), which interacts with MinD and inhibits the bundling of FtsZ filaments.

S1 pocket of glutamate carboxypeptidase II: a new binding site for amyloid-β degradation.

We recently reported that glutamate carboxypeptidase II (GCPII) has a new physiological function degrading amyloid-β (Aβ), distinct from its own hydrolysis activity in N-acetyl-L-aspartyl-L-glutamate (NAAG); however, its underlying mechanism remains undiscovered. Using site-directed mutagenesis and S1 pocket-specific chemical inhibitor (compound 2), which was developed for the present study based on in sillico computational modeling, we discovered that the Aβ degradation occurs through S1 pocket but not through S1' pocket responsible for NAAG hydrolysis. Treatment with compound 2 prevented GCPII from Aβ degradation without any impairment in NAAG hydrolysis.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence.

Crystallization and preliminary X-ray crystallographic analysis of sterol transcription factor Upc2 from Saccharomyces cerevisiae.

Upc2, a zinc-cluster transcription factor, is a regulator of ergosterol biosynthesis in yeast. In response to sterol levels, the transcriptional activity of Upc2 is controlled by the C-terminal domain. In this study, the C-terminal regulatory domain of Upc2 from Saccharomyces cerevisiae was purified and crystallized by the vapour-diffusion method.

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution.

Localization of a site of action for benzofuroindole-induced potentiation of BKCa channels.

As previously reported, the activity of the large-conductance calcium (Ca(2+))-activated potassium (K(+)) (BK(Ca)) channel is strongly potentiated from the extracellular side of the cell membrane by certain benzofuroindole derivatives. Here, the mechanism of action of one of the most potent activators, 4-chloro-7-(trifluoromethyl)-10H-benzofuro[3,2-b]indole-1-carboxylic acid (CTBIC), is characterized. This compound, Compound 22 in the previous report (Chembiochem 6:1745-1748, 2005), potentiated the activity of the channel by shifting its conductance-voltage relationship toward the more negative direction.

Purification, crystallization and preliminary X-ray crystallographic analysis of a methanol dehydrogenase from the marine bacterium Methylophaga aminisulfidivorans MP(T).

Methylophaga aminisulfidivorans MP(T) is a marine methylotrophic bacterium that utilizes C(1) compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c(L).

Design, synthesis and X-ray crystallographic study of NAmPRTase inhibitors as anti-cancer agents.

NAmPRTase (PBEF/Visfatin) plays a pivotal role in the salvage pathway of NAD(+) biosynthesis. NAmPRTase has been an attractive target for anti-cancer agents that induce apoptosis of tumor cells via a declining plasma NAD(+) level. In this report, a series of structural analogs of FK866 (1), a known NAmPRTase inhibitor, was synthesized and tested for inhibitory activities against the proliferation of cancer cells and human NAmPRTase.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.

Ryanodine receptor assembly: a novel systems biology approach to 3D mapping.

Ryanodine receptors (RyRs) are intracellular Ca(2+) release channels (CRCs) that play a pivotal role in cellular Ca(2+) signaling. In striated muscles, RyR-mediated Ca(2+) release from the sarcoplasmic reticulum (SR) induces elevation of cytosolic Ca(2+) concentration and subsequent muscle contraction. Evidence from various sources suggests that RyRs in homo-tetrameric conformation form a large conductance Ca(2+) permeable channel in the central pore and large cytoplasmic domains.

Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice.

Background: Oxidative stress is imperative for its morbidity towards diabetic complications, where abnormal metabolic milieu as a result of hyperglycemia, leads to the onset of several complications. A biological antioxidant capable of inhibiting oxidative stress mediated diabetic progressions; during hyperglycemia is still the need of the era. The current study was performed to study the effect of biologically synthesized gold nanoparticles (AuNPs) to control the hyperglycemic conditions in streptozotocin induced diabetic mice.

Crystallization and preliminary X-ray crystallographic analysis of MinE, the cell-division topological specificity factor from Helicobacter pylori.

Cell division in Gram-negative bacteria is driven by the formation of an FtsZ ring at the division site. MinE regulates the proper placement of the FtsZ ring at mid-cell by blocking the inhibitory action of the MinCD complex. Diffraction data were collected at 2.

Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor.

In Gram-negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD).