Alexey D. Nikulin

Structure of Pseudomonas aeruginosa Hfq protein.

The structure of the Hfq protein from Pseudomonas aeruginosa was determined using two different ionic conditions. In both cases the molecules formed identical hexameric rings, but some variations in the crystal packing were revealed. Hfq belongs to the family of Sm/LSm proteins, the members of which can form hexameric as well as heptameric rings. Comparative analysis of known structures of this protein family shows that the fragment of the Sm-fold responsible for oligomerization is strongly structurally conserved. In the heptameric ring, three conserved hydrogen bonds between beta-strands of adjacent molecules hold together the monomers, whereas in the hexameric rings of Hfq an additional conserved inaccessible hydrogen bond between neighbouring monomers is observed.

The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance

BACKGROUND . The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA. L22 has been found on the cytoplasmic side of the 50S ribosomal subunit. It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known. Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome. RESULTS . The structure of Thermus thermophilus L22 was solved using X-ray crystallography. L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long. A large part of the surface area of the protein has the potential to be involved in interactions with rRNA. A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin. CONCLUSIONS . The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA. The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions. This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm.

Structure of Citrobacter freundii l-methionine γ-­lyase

l-Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes γ-elimination of l-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.

Hfq binds ribonucleotides in three different RNA-binding sites.

The Hfq protein forms a doughnut-shaped homohexamer that possesses RNA-binding activity. There are two distinct RNA-binding surfaces located on the proximal and the distal sides of the hexamer. The proximal side is involved in the binding of mRNA and small noncoding RNAs (sRNAs), while the distal side has an affinity for A-rich RNA sequences. In this work, the ability of various ribonucleotides to form complexes with Hfq from Pseudomonas aeruginosa has been tested using X-ray crystallography. ATP and ADPNP have been located in the distal R-site, which is a site for poly(A) RNA binding. UTP has been found in the so-called lateral RNA-binding site at the proximal surface. CTP has been found in both the distal R-site and the proximal U-binding site. GTP did not form a complex with Hfq under the conditions tested. The results have demonstrated the power of the crystallographic method for locating ribonucleotides and predicting single-stranded RNA-binding sites on the protein surface.

Formation of amyloid-like fibrils by Y-box binding protein 1 (YB-1) is mediated by its cold shock domain and modulated by disordered terminal domains.

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions.

Structure of the ribosomal protein L1-mRNA complex at 2.1 A resolution: common features of crystal packing of L1-RNA complexes.

The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1 A resolution. It was found that all polar atoms involved in conserved protein-RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K(+) ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1-RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open-open or open-closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation.

RNA-binding Sm-like proteins of bacteria and archaea. Similarity and difference in structure and function

RNA-binding proteins play a significant role in many processes of RNA metabolism, such as splicing and processing, regulation of DNA transcription and RNA translation, etc. Among the great number of RNA-binding proteins, so-called RNA-chaperones occupy an individual niche; they were named for their ability to assist RNA molecules to gain their accurate native spatial structure. When binding with RNAs, they possess the capability of altering (melting) their secondary structure, thus providing a possibility for formation of necessary intramolecular contacts between individual RNA sites for proper folding. These proteins also have an additional helper function in RNA-RNA and RNA-protein interactions. Members of such class of the RNA-binding protein family are Sm and Sm-like proteins (Sm-Like, LSm). The presence of these proteins in bacteria, archaea, and eukaryotes emphasizes their biological significance. These proteins are now attractive for researchers because of their implication in many processes associated with RNAs in bacterial and archaeal cells. This review is focused on a comparison of architecture of bacterial and archaeal LSm proteins and their interaction with different RNA molecules.

Studies of Polymorphism of Amyloid-β 42 Peptide from Different Suppliers

The aim of this study was to investigate the process of amyloidogenesis of amyloid-β (Aβ)42 peptide, by means of fluorescence spectroscopy, electron microscopy, X-ray diffraction, and mass spectrometry. It has been repeatedly reported in the literature that the process of fibril formation by Aβ42 peptide depends considerably not only upon the specific conditions (ionic conditions, pH, temperature, mixing, etc.), as well as the manufacturing route (synthetic or recombinant), but also on the methods of synthesis and purification. We have, for the first time, systematically analyzed samples of Aβ42 peptide supplied by five different companies (Anaspec, Invitrogen, Enzo, Sigma-Aldrich, and SynthAssist) and obtained evidence of significant variability, including lot to lot variations. All studied samples formed amyloid-like fibrils at pH3-6, and the fibrils contained cross-β structures. Samples from Anaspec, Invitrogen, and Enzo formed one particular type of amyloid-like fibrils, while the samples from Sigma-Aldrich and SynthAssist formed another distinct type of fibrils. The observed polymorphism emphasizes the capacity of the Aβ42 peptide to act as a prion agent with varying structural characteristics. The presented data have allowed us to propose a possible mechanism of formation of amyloid-like fibrils.

Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates

Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ-(L-1-amino-3-methylthiopropylphosphinic acid) and β-(S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-nor-leucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5′-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

Effect of Conserved Intersubunit Amino Acid Substitutions on Hfq Protein Structure and Stability

Hfq is a thermostable RNA-binding bacterial protein that forms a uniquely shaped homohexamer. Based on sequence and structural similarity, Hfq belongs to the like-Sm (LSm) protein family. In spite of a rather high degree of homology between archaeal and eukaryotic LSm proteins, their quaternary structure is different, usually consisting of five to eight monomers. In this work, the importance of conserved intersubunit hydrogen bonds for the Hfq spatial organization was tested. The structures and stabilities for the Gln8Ala, Asn28Ala, Asp40Ala, and Tyr55Ala Hfq mutants were determined. All these proteins have the same hexamer organization, but their stability is different. Elimination of a single intersubunit hydrogen bond due to Gln8Ala, Asp40Ala, and Tyr55Ala substitutions results in decreased stability of the Hfq hexamer. Tyr55Ala Hfq as well as the earlier studied His57Ala Hfq has reduced protein thermostability, which seems to correspond to an opening of the protein hydrophobic core.