ITS1 variation among Stichopathes cf. maldivensis (Hexacorallia: Antipatharia) whip black corals unveils conspecificity and population connectivity at local and global scales across the Indo-Pacific

Coral Reefs - Due to the dearth of molecular markers variable enough to distinguish species of black corals, species delimitation in Antipatharia is still mainly based on morphological traits. One...     

Structural processes during starch granule hydration by synchrotron radiation microdiffraction

Starch granule hydration has been examined on the level of a single potato starch granule by static and dynamic synchrotron radiation (SR) microdiffraction techniques. A cryofrozen, hydrated granule was mapped through a 5 microm SR-beam in order to investigate its internal organization. The edge of the granule showed fiber texture scattering due to radially oriented amylopectin helices. The variation of fiber texture across the granule center supports the model of concentric shells. The crystalline phase appears, however, to increase strongly toward the granule center due to a random amylopectin fraction, which could be related to crystallization of short-range ordered amylopectin during hydration. During gelatinization, the shell structure breaks down and remaining fiber-textured amylopectin domains belong probably to the swollen starch granule envelope. Hydration of a granule was initiated by a microdrop generator and followed in situ by SR-microdiffraction. A fast hydration process with a half time of about 7 s seems to reflect the porous nature of starch granules. The size of the hydrated domains suggests that this process is limited to the level of amylopectin side chain clusters. Longer hydration times are assumed to involve remaining short-range ordered amylopectin and results in larger domains.     

The crystal structure of the extracellular domain of the inhibitor receptor expressed on myeloid cells IREM-1

The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands.     

The mitochondrial genome of Pocillopora (Cnidaria: Scleractinia) contains two variable regions: the putative D-loop and a novel ORF of unknown function

The complete mitochondrial genomes of two individuals attributed to different morphospecies of the scleractinian coral genus Pocillopora have been sequenced. Both genomes, respectively 17,415 and 17,422 nt long, share the presence of a previously undescribed ORF encoding a putative protein made up of 302 amino acids and of unknown function. Surprisingly, this ORF turns out to be the second most variable region of the mitochondrial genome (1% nucleotide sequence difference between the two individuals) after the putative control region (1.5% sequence difference). Except for the presence of this ORF and for the location of the putative control region, the mitochondrial genome of Pocillopora is organized in a fashion similar to the other scleractinian coral genomes published to date. For the first time in a cnidarian, a putative second origin of replication is described based on its secondary structure similar to the stem-loop structure of O(L), the origin of L-strand replication in vertebrates.     

Structuring detergents for extracting and stabilizing functional membrane proteins

Background

Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.

Methodology/Principal Findings

Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by ¹H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.

Conclusion/Significance

These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.     

Do cryptic species matter in macroecology? Sequencing European groundwater crustaceans yields smaller ranges but does not challenge biodiversity determinants

Ecologists increasingly rely on molecular delimitation methods (MMs) to identify species boundaries, thereby potentially increasing the number of putative species because of the presence of morphologically cryptic species. It has been argued that cryptic species could challenge our understanding of what determine large‐scale biodiversity patterns which have traditionally been documented from morphology alone. Here, we used morphology and three MMs to derive four different sets of putative species among the European groundwater crustaceans. Then, we used regression models to compare the relative importance of spatial heterogeneity, productivity and historical climates, in shaping species richness and range size patterns across sets of putative species. We tested three predictions. First, MMs would yield many more putative species than morphology because groundwater is a constraining environment allowing little morphological changes. Second, for species richness, MMs would increase the importance of spatial heterogeneity because cryptic species are more likely along physical barriers separating ecologically similar regions than along resource gradients promoting ecologically‐based divergent selection. Third, for range size, MMs would increase the importance of historical climates because of reduced and asymmetrical fragmentation of large morphological species ranges at northern latitudes. MMs yielded twice more putative species than morphology and decreased by 10‐fold the average species range size. Yet, MMs strengthened the mid‐latitude ridge of high species richness and the Rapoport effect of increasing range size at higher latitudes. Species richness predictors did not vary between morphology and MMs but the latter increased the proportion of variance in range size explained by historical climates. These findings demonstrate that our knowledge of groundwater biodiversity determinants is robust to overlooked cryptic species because the latter are homogeneously distributed along environmental gradients. Yet, our findings call for incorporating multiple species delimitation methods into the analysis of large‐scale biodiversity patterns across a range of taxa and ecosystems.     

Fast intracrystalline hydration of beta-chitin revealed by combined microdrop generation and on-line synchrotron radiation microdiffraction

Water microdrops of about 50 microm in diameter, generated by an ink-jet system, have been used to hydrate fragments of Pogonophora tubes. In situ X-ray microdiffraction with a beam size of 10 microm was used to follow the structural transformations that affected the crystalline beta-chitin part of the specimens. Starting from anhydrous chitin, the formation of a full beta-chitin dihydrate was observed within about 90 s. A disordered intermediate phase with variable d-spacing that could be due to a mixture of anhydrous and hydrated beta-chitin layers was also detected.     

A Bridge Crosses the Active-Site Canyon of the Epstein-Barr Virus Nuclease with DNase and RNase Activities

Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn²⁺, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.     

Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system

The pathogenic bacterium Shigella flexneri uses a type III secretion system to inject virulence factors from the bacterial cytosol directly into host cells. The machinery that identifies secretion substrates and controls the export of extracellular components and effector proteins consists of several inner-membrane and cytoplasmic proteins. One of the inner membrane components, Spa40, belongs to a family of proteins proposed to regulate the switching of substrate specificity of the export apparatus. We show that Spa40 is cleaved within the strictly conserved amino acid sequence NPTH and substitution of the proposed autocatalytic residue abolishes cleavage. Here we also report the crystal structure of the cytoplasmic complex Spa40_C and compare it with the recent structures of the homologues from Escherichia coli and Salmonella typhimurium . These structures reveal the tight association of the cleaved fragments and show that the conserved NPTH sequence lies on a loop which, when cleaved, swings away from the catalytic N257 residue, resulting in different surface features in this region. This structural rearrangement suggests a mechanism by which non-cleaving forms of these proteins interfere with correct substrate switching of the apparatus.