Splicing Site Recognition by Synergy of Three Domains in Splicing Factor RBM10.

Splicing factor RBM10 and its close homologues RBM5 and RBM6 govern the splicing of oncogenes such as Fas, NUMB, and Bcl-X. The molecular architecture of these proteins includes zinc fingers (ZnFs) and RNA recognition motifs (RRMs). Three of these domains in RBM10 that constitute the RNA binding part of this splicing factor were found to individually bind RNAs with micromolar affinities.

Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding.

The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in double-stranded DNA (dsDNA). Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and molecular dynamics simulations of homologous homeodomain-DNA complexes. In contrast, our nuclear magnetic resonance (NMR) studies show that DNA binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17-Arg52 salt bridge.

NMR in structural genomics to increase structural coverage of the protein universe: Delivered by Prof. Kurt Wüthrich on 7 July 2013 at the 38th FEBS Congress in St. Petersburg, Russia.

For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe.

Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif.

Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism.

UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported.

Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome.

The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.

NMR reveals structural rearrangements associated to substrate insertion in nucleotide-adding enzymes.

The protein NP_344798.1 from Streptococcus pneumoniae TIGR4 exhibits a head and base-interacting neck domain architecture, as observed in class II nucleotide-adding enzymes. Although it has less than 20% overall sequence identity with any member of this enzyme family, the residues involved in substrate-recognition and catalysis are highly conserved in NP_344798.

Nuclear Magnetic Resonance Structure of a Novel Globular Domain in RBM10 Containing OCRE, the Octamer Repeat Sequence Motif.

The OCtamer REpeat (OCRE) has been annotated as a 42-residue sequence motif with 12 tyrosine residues in the spliceosome trans-regulatory elements RBM5 and RBM10 (RBM [RNA-binding motif]), which are known to regulate alternative splicing of Fas and Bcl-x pre-mRNA transcripts. Nuclear magnetic resonance structure determination showed that the RBM10 OCRE sequence motif is part of a 55-residue globular domain containing 16 aromatic amino acids, which consists of an anti-parallel arrangement of six β strands, with the first five strands containing complete or incomplete Tyr triplets. This OCRE globular domain is a distinctive component of RBM10 and is more widely conserved in RBM10s across the animal kingdom than the ubiquitous RNA recognition components.

Cofactor-induced reversible folding of Flavodoxin-4 from Lactobacillus acidophilus.

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN-binding mechanism to flavodoxins-4 were obtained from the NMR structures of the apo-protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN.

NMR structure determination of the protein NP_344798.1 as the first representative of Pfam PF06042.

We present the NMR structure determination of the protein NP_344798.1, which forms a CCA-adding enzyme head-domain architecture and is the first structural representative of the Pfam protein family PF06042. Its structure can now serve as a template for homology modeling of the other 785 members of this protein family.

J-UNIO protocol used for NMR structure determination of the 206-residue protein NP_346487.1 from Streptococcus pneumoniae TIGR4.

The NMR structure of the 206-residue protein NP_346487.1 was determined with the J-UNIO protocol, which includes extensive automation of the structure determination. With input from three APSY-NMR experiments, UNIO-MATCH automatically yielded 77 % of the backbone assignments, which were interactively validated and extended to 97 %.

APSY-NMR for protein backbone assignment in high-throughput structural biology.

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h.

NMR structures of α-proteobacterial ATPase-regulating ζ-subunits.

NMR structures of ζ-subunits, which are recently discovered α-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ζ-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ζ-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in α-proteobacteria.

NMR-profiles of protein solutions.

NMR-Profiles are quantitative one-dimensional (1D) presentations of 2D [¹⁵N, ¹H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, an NMR-Profile is recorded at the outset of a structure determination, using a uniformly ¹⁵N-labeled microscale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR.

Structural representative of the protein family PF14466 has a new fold and establishes links with the C2 and PLAT domains from the widely distant Pfams PF00168 and PF01477.

The domain of unknown function (DUF) YP_001302112.1, a protein secreted by the human intestinal microbita, has been determined by NMR and represents the first structure for the Pfam PF14466. Its NMR structure is classified as a new fold, which, nonetheless, shows limited similarities with representatives of the PLAT/LH2 domains from PF01477 and the C2 domains from PF00168, both of which bind Ca(2+) for their physiological functions.

Thermodynamic stability of psychrophilic and mesophilic pheromones of the protozoan ciliate euplotes.

Three psychrophilic protein pheromones (En-1, En-2 and En-6) from the polar ciliate, Euplotes nobilii, and six mesophilic pheromones (Er-1, Er-2, Er-10, Er-11, Er-22 and Er-23) from the temperate-water sister species, Euplotes raikovi, were studied in aqueous solution for their thermal unfolding and refolding based on the temperature dependence of their circular dichroism (CD) spectra. The three psychrophilic proteins showed thermal unfolding with mid points in the temperature range 55-70 °C. In contrast, no unfolding was observed for any of the six mesophilic proteins and their regular secondary structures were maintained up to 95 °C.

The J-UNIO protocol for automated protein structure determination by NMR in solution.

The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of APSY-NMR spectroscopy, 3D heteronuclear-resolved [(1)H,(1)H]-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient.

NMR structure of the Bordetella bronchiseptica protein NP_888769.1 establishes a new phage-related protein family PF13554.

The solution structure of the hypothetical phage-related protein NP_888769.1 from the Gram-negative bacterium Bordetella bronchoseptica contains a well-structured core comprising a five-stranded, antiparallel β-sheet packed on one side against two α-helices and a short β-hairpin with three flexibly disordered loops extending from the central β-sheet. A homology search with the software DALI identified two Protein Data Bank deposits with Z-scores > 8, where both of these proteins have less than 8% sequence identity relative to NP_888769.

Comparison of NMR and crystal structures for the proteins TM1112 and TM1367.

The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.

NMR structure of the protein NP_247299.1: comparison with the crystal structure.

The NMR structure of the protein NP_247299.1 in solution at 313 K has been determined and is compared with the X-ray crystal structure, which was also solved in the Joint Center for Structural Genomics (JCSG) at 100 K and at 1.7 Å resolution.

Microscale NMR screening of new detergents for membrane protein structural biology.

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E.

Novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus.

The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel alpha/beta-fold formed by a mixed parallel/antiparallel six-stranded beta-barrel, an alpha-helix covering one opening of the barrel, and a 3(10)-helix alongside the barrel.

Genome-wide insertional mutagenesis of Arabidopsis thaliana.

Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels.