Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition

Recognition of the 3′-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3′-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA comple

The Ginger-shaped Asteroid 4179 Toutatis: New Observations from a Successful Flyby of Chang'e-2

On 13 December 2012, Chang'e-2 conducted a successful flyby of the near-Earth asteroid 4179 Toutatis at a closest distance of 770 $\pm$ 120 meters from the asteroid's surface. The highest-resolution image, with a resolution of better than 3 meters, reveals new discoveries on the asteroid, e.g., a giant basin at the big end, a sharply perpendicular silhouette near the neck region, and direct evidence of boulders and regolith, which suggests that Toutatis may bear a rubble-pile structure. Toutatis' maximum physical length and width are (4.75 $\times$ 1.95 km) $\pm$10$\%$, respectively, and the direction of the +$z$ axis is estimated to be (250$\pm$5$^\circ$, 63$\pm$5$^\circ$) with respect to the J2000 ecliptic coordinate system. The bifurcated configuration is indicative of a contact binary origin for Toutatis, which is composed of two lobes (head and body). Chang'e-2 observations have significantly improved our understanding of the characteristics, formation, and evolution of asteroids in general.Comment: 21 pages, 3 figures, 1 tabl

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli

The haem-binding protein HbpS from Streptomyces reticuli was crystallized and diffraction data were collected to a maximal resolution of 2.25 Å

Enhancing functional expression of L-glycerophosphate oxidase in Escherichia coli by controlling the expression rate

Heterologous expression of proteins often pursues high expression levels, but it can easily result in misfolding and loss of biological function. L-α-glycerophosphate oxidase (GlpO) is a flavin adenine dinucleotide (FAD)-dependent oxidase which is widely used in the clinical determination of triglycerides. We found that the total enzymatic activity of GlpO expressed in Escherichia coli (E. coli) was extremely low, probably due to the absence of FAD cofactors and the misfolding of GlpO at a high synthesis rate. Therefore, decreasing the expression rate was used to improve the activity of GlpO. The specific activity of GlpO expressed on the pUC19 vector with lac promotor was approximately 30 times higher than that expressed on the pET28a vector with T7 promotor, but the expression levels of GlpO on the two vectors were completely opposite. It indicated that the specific activity of GlpO was increased as the expression level decreased. However, too low expression greatly influences the total amount and activity of the functional enzyme. In order to resolve this problem, two new plasmids, GlpO-CG4 and GlpO-CG6, were constructed by inserting 4 or 6 nucleotides, respectively, between the ribosome binding site (RBS) and the start code (ATG) on pET28a. Compared with the expression on the GlpO-pET vector, the expression rates of GlpO on the GlpO-CG4 and GlpO-CG6 were dramatically decreased. The total activity of GlpO expressed on GlpO-CG6 was 11 times and 1.5 times higher than that expressed on the GlpO-pET and GlpO-pUC, respectively. Results suggest that the activity of GlpO can be improved by decreasing the expression rate.&nbsp

Structural and functional analysis of the DEAF-1 and BS69 MYND domains.

DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a ββα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins