Secondary structure of the r(CUUCGG) tetraloop.

The oligonucleotide r(GGACUUCGGUCC) has been observed to adopt a hairpin conformation by solution NMR and a double helical conformation by X-ray diffraction. In order to understand this apparent conflict, we used time-resolved fluorescence depolarization and 19fluorine NMR to follow the secondary structure of this dodecamer as the solution composition was changed stepwise from the NMR experimental conditions to those used for crystallization. Calculation of the dodecamer concentration in the crystal (180 mM strands) and the cation concentration needed for neutrality (>2 M) prompted investigation of a tethered species, in which two dodecamers are connected by a string of 4 nt, geometrically equivalent to approximately 100 mM strands, in 2.5 M NaCl. The RNA tetraloop and its DNA analog maintain a single-strand hairpin conformation in solution, even under the conditions used to grow the crystal. Under high salt conditions, the tethered RNA and DNA analogs of this sequence yield secondary components which could be the double helical conformation. Crystal contacts in addition to solvent changes and high RNA concentrations are needed to obtain the double helix as the predominant species.     

Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A₂, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (V_max∼15.5 μmol/min/mg; K_m∼83 μM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall–deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.     

Biological recognition of phosphate and sulfate

The biological recognition of trigonal pyramids has been studied in a statistical analysis of the Cambridge Structural Data Base. The preferential stereochemistry of pyramidal anion-Lewis acid interactions is easily described for the phosphonyl dianion (R-PO3(2-), as found in the phosphate group) and the sulfonyl monoanion (R-SO3-, as found in the sulfate group) by trans/gauche conformational terminology. Interactions between these pyramidal anions and metals generally prefer gauche geometry (as defined by the R-X = O-Mn+ torsion angle, X = P or S). Interactions between phosphonyls and hydrogen bond donors likewise display a preference for gauche orientation. Interactions between sulfonyls and hydrogen bond donors exhibit a preference for gauche stereochemistry and also cluster in eclipsed regions. These hydrogen bond motifs provide a greater understanding of the function of pyramidal anions in biological structure and function. For example, interactions of the sulfate monoanion are important for the binding of heparin, a sulfated glycosaminoglycan, to antithrombin III.     

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer''s disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC₅₀ of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.     

The Synthesis of Oligonucleotides Containing Fluoro-2′-Deoxycytidine for Secondary Structure Determination of Tandem Tetraloop DNA Analogs

Abstract

A method to convert 5-FdU phosphoramidite to 4-triazol-5- FdU phosphoramidite was used to make deoxyoligonucleotides containing 5- FdC to follow a rare transition between a tandem hairpin and a single hairpin form, which results in moving a distant 5-FdC, 5-FdU bases into juxtaposition.     

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling

To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.     

Crystallization and oligomeric structure of rat liver arginase.

Rat liver arginase, a manganese-metalloenzyme, has been crystallized from polyethylene glycol 8000 in N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer at pH 8.5. Crystals form as either cubes or pyramids and belong to space group P3(1) (or P3(2)) with hexagonal unit cell dimensions a = b = 88.9 A, c = 114.8 A, or a = b = 88.5 A, c = 104.5 A; the variation along the c axis does not correlate with the external crystal morphology of cube or pyramid-shaped. X-ray diffraction data are measured to a limiting resolution of 2.4 A. Given the volume constraints of the unit cell it is likely that rat liver arginase is a trimer, with three 35,000 Da monomers in the asymmetric unit. This resolves a persistent ambiguity regarding the oligomeric structure of this enzyme.     

Antibody binding defines a structure for an epitope that participates in the PrPC-->PrPSc conformational change.

The X-ray crystallographic structures of the anti-Syrian hamster prion protein (SHaPrP) monoclonal Fab 3F4 alone, as well as the complex with its cognate peptide epitope (SHaPrP 104-113), have been determined to atomic resolution. The conformation of the decapeptide is an Omega-loop. There are substantial alterations in the antibody combining region upon epitope binding. The peptide binds in a U-shaped groove on the Fab surface, with the two specificity determinants, Met109 and Met112, penetrating deeply into separate hydrophobic cavities formed by the heavy and light chain complementarity-determining regions. In addition to the numerous contacts between the Fab and the peptide, two intrapeptide hydrogen bonds are observed, perhaps indicating the structure bound to the Fab exists transiently in solution. This provides the first structural information on a portion of the PrP N-terminal region observed to be flexible in the NMR studies of SHPrP 90-231, SHaPrP 29-231 and mouse PrP 23-231. Antibody characterization of the antigenic surfaces of PrPC and PrPSc identifies this flexible region as a component of the conformational rearrangement that is an essential feature of prion disease.     

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.     

Crystal structures of Bacillus caldovelox arginase in complex with substrate and inhibitors reveal new insights into activation, inhibition and catalysis in the arginase superfamily

BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.