Crystal structures of PI3K-C2α PX domain indicate conformational change associated with ligand binding

<p>Abstract</p> <p>Background</p> <p>PX domains have specialized protein structures involved in binding of phosphoinositides (PIs). Through binding to the various PIs PX domains provide site-specific membrane signals to modulate the intracellular localisation and biological activity of effector proteins. Several crystal structures of these domains are now available from a variety of proteins. All PX domains contain a canonical core structure with main differences exhibited within the loop regions forming the phosphoinositide binding pockets. It is within these areas that the molecular basis for ligand specificity originates.</p> <p>Results</p> <p>We now report two new structures of PI3K-C2α PX domain that crystallised in a P3₁21 space group. The two structures, refined to 2.1 Å and 2.5 Å, exhibit significantly different conformations of the phosphoinositide-binding loops. Unexpectedly, in one of the structures, we have detected a putative-ligand trapped in the binding site during the process of protein purification and crystallisation.</p> <p>Conclusion</p> <p>The two structures reported here provide a more complete description of the phosphoinositide binding region compared to the previously reported 2.6 Å crystal structure of human PI3K-C2α PX where this region was highly disordered. The structures enabled us to further analyse PI specificity and to postulate that the observed conformational change could be related to ligand-binding.</p

Retired A Stars and Their Companions: Exoplanets Orbiting Three Intermediate-Mass Subgiants

We report precision Doppler measurements of three intermediate-mass subgiants from Lick and Keck Observatories. All three stars show variability in their radial velocities consistent with planet-mass companions in Keplerian orbits. We find a planet with a minimum mass of 2.5 Mjup in a 351.5 day orbit around HD 192699, a planet with a minimum mass of 2.0 Mjup in a 341.1 day orbit around HD 210702, and a planet with a minimum mass of 0.61 Mjup in a 297.3 day orbit around HD 175541. Stellar mass estimates from evolutionary models indicate that all of these stars were formerly A-type dwarfs with masses ranging from 1.65 to 1.85 Msun. These three long-period planets would not have been detectable during their stars' main-sequence phases due to the large rotational velocities and stellar jitter exhibited by early-type dwarfs. There are now 9 "retired" (evolved) A-type stars (Mstar > 1.6 Msun) with known planets. All 9 planets orbit at distances a \geq 0.78 AU, which is significantly different than the semimajor axis distribution of planets around lower-mass stars. We examine the possibility that the observed lack of close-in planets is due to engulfment by their expanding host stars, but we find that this explanation is inadequate given the relatively small stellar radii of K giants (Rstar < 32 Rsun = 0.15 AU) and subgiants (Rstar < 7 Rsun = 0.03 AU). Instead, we conclude that planets around intermediate-mass stars reside preferentially beyond ~0.8 AU, which may be a reflection of different formation and migration histories of planets around A-type stars.Comment: 31 pages, 9 figures, 6 tables, ApJ accepted, corrected minor typo

Nonstomatal limitations are responsible for drought-induced photosynthetic inhibition in four C4 grasses

Here, the contribution of stomatal and nonstomatal factors to photosynthetic inhibition under water stress in four tropical C(4) grasses was investigated (Panicum coloratum, Bothriochloa bladhii, Cenchrus ciliaris and Astrebla lappacea ). Plants were grown in well watered soil, and then the effects of soil drying were measured on leaf gas exchange, chlorophyll a fluorescence and water relations. During the drying cycle, leaf water potential (Psi(leaf)) and relative water content (RWC) decreased from c. -0.4 to -2.8 MPa and 100-40%, respectively. The CO(2) assimilation rates (A) and quantum yield of PSII (Phi(PSII)) of all four grasses decreased rapidly with declining RWC. High CO(2) concentration (2500 mul l(-1)) had no effect on A or Phi(PSII) at any stage of the drying cycle. Electron transport capacity and dark respiration rates were unaltered by drought. The CO(2) compensation concentrations of P. coloratum and C. ciliaris rose sharply when leaf RWC fell below 70%. In P. coloratum, 5% CO(2) did not prevent the decline of O(2) evolution rates under water stress. We conclude that inhibition of photosynthesis in the four C(4) grasses under water stress is dependent mainly on biochemical limitations

SdPI, The First Functionally Characterized Kunitz-Type Trypsin Inhibitor from Scorpion Venom

Background: Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized. Principal Findings: A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (Ki = 1.6610 27 M) and thermostability. Conclusions: The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitztype venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derive

NMR and mutational identification of the collagen-binding site of the chaperone Hsp47.

Heat shock protein 47 (Hsp47) acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective (15)N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases

Estimated Costs of False Laboratory Diagnoses of Tuberculosis in Three Patients

We estimated direct medical and nonmedical costs associated with a false diagnosis of tuberculosis (TB) caused by laboratory cross-contamination of Mycobacterium tuberculosis cultures in Massachusetts in 1998 and 1999. For three patients who received misdiagnoses of active TB disease on the basis of laboratory cross-contamination, the costs totaled U.S.$32,618. Of the total, 97$10,873 (range, $1,033-$21,306). Reducing laboratory cross-contamination and quickly identifying patients with cross-contaminated cultures can prevent unnecessary and potentially dangerous treatment regimens and anguish for the patient and financial burden to the health-care system