Alexei Nikulin

Structure of the L1 protuberance in the ribosome.

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 Å resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1–rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.

Crystal structure of the S15-rRNA complex.

In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 Å resolution crystal structure of the highly conserved S15–rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.

The structures of mutant forms of Hfq from Pseudomonas aeruginosa reveal the importance of the conserved His57 for the protein hexamer organization.

The bacterial Sm-like protein Hfq forms homohexamers both in solution and in crystals. The monomers are organized as a continuous beta-sheet passing through the whole hexamer ring with a common hydrophobic core. Analysis of the Pseudomonas aeruginosa Hfq (PaeHfq) hexamer structure suggested that solvent-inaccessible intermonomer hydrogen bonds created by conserved amino-acid residues should also stabilize the quaternary structure of the protein. In this work, one such conserved residue, His57, in PaeHfq was replaced by alanine, threonine or asparagine. The crystal structures of His57Thr and His57Ala Hfq were determined and the stabilities of all of the mutant forms and of the wild-type protein were measured. The results obtained demonstrate the great importance of solvent-inaccessible conserved hydrogen bonds between the Hfq monomers in stabilization of the hexamer structure.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Molecular mimicry in translational regulation: the case of ribosomal protein S15.

Ribosomal protein S15 is highly conserved among prokaryotes. It plays a pivotal role in the assembly of the central domain of the small ribosomal subunit and regulates its own expression by a feedback mechanism at the translational level. The protein recognizes two RNA targets (rRNA and mRNA) that share only partial similarity. Its interaction with 16S rRNA has been fully characterized, while mRNA interactions and regulatory mechanisms have been extensively studied in E. coli and in T. thermophilus. Recently, we have characterized which aminoacids are involved in E. coli mRNA recognition, using an in vivo assay allowing to identify S15 mutations affecting the S15-mRNA interactions without altering 30S subunit assembly. Here, we address the following questions: Are common determinants used by S15 to recognize its rRNA and mRNA targets? What is the extent of molecular mimicry? Is the regulatory mechanism conserved? Our results indicate that specific recognition of mRNA and rRNA relies on both mimicry and site differentiation. They also highlight the high plasticity of RNA to adapt to evolutionary constraints.

Domain II of Thermus thermophilus Ribosomal Protein L1 Hinders Recognition of Its mRNA

The two-domain ribosomal protein L1 has a dual function as a primary rRNA-binding ribosomal protein and as a translational repressor that binds its own mRNA. Here, we report the crystal structure of a complex between the isolated domain I of L1 from the bacterium Thermus thermophilus and a specific mRNA fragment from Methanoccocus vannielii. In parallel, we report kinetic characteristics measured for complexes formed by intact TthL1 and its domain I with the specific mRNA fragment. Although, there is a close similarity between the RNA-protein contact regions in both complexes, the association rate constant is higher in the case of the complex formed by the isolated domain I. This finding demonstrates that domain II hinders mRNA recognition by the intact TthL1.

STRUCTURAL STUDIES OF RIBOSOMAL PROTEINS

Crystal and solution structures of fourteen ribosomal proteins from thermophilic bacteria have been determined during the last decade. This paper reviews structural studies of ribosomal proteins from Thermus thermophilus carried out at the Institute of Protein Research (Pushchino, Russia) in collaboration with the University of Lund (Lund, Sweden) and the Center of Structural Biochemistry (Karolinska Institute, Huddinge, Sweden). New experimental data on the crystal structure of the ribosomal protein L30 from T. thermophilus are also included.

Crystal Structure of the Archaeal Translation Initiation Factor 2 in Complex with a GTP Analogue and Met-tRNAfMet

Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation initiation factor 2) in its GTP-bound form delivers Met-tRNAi(Met) to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNAi. Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ⋅GDPNP⋅Met-tRNAf(Met) has been solved at 3.2Å resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-Å-resolution structure of almost the same ternary initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Background: Speculative chemical mechanism of methionine γ-lyase is formulated, kinetic and structural data concerning elementary stages of physiological reaction are mostly lacking. Results: Pre-steady-state kinetic and structural analysis of the enzyme interaction with inhibitors was performed. Conclusion: Results elucidate the mechanism of intermediate interconversion at initial stages of enzymatic reaction. Significance: The data serve for understanding detailed mechanism of pyridoxal 5′-phosphate-dependent γ-elimination reaction. Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.