Recognition of γ-Subunit by ß-Subunit in Translation Initiation Factor 2. Stabilization of the GTP-Bound State of I/F 2 in Archaea and Eukaryotes.

Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) functions as a heterotrimeric complex. It consists of three subunits (α, ß, γ). α- and ß-subunits are bound to γ-subunit by hydrogen bonds and van der Waals interactions, but do not contact each other. Although main functions of the factor are performed by the γ-subunit, reliable formation of αγ and ßγ complexes is necessary for its proper functioning. In this work, we introduced mutations in the recognition part of the ßγ interface and showed that hydrophobic effect plays a crucial role in the recognition of subunits both in eukaryotes and archaea. Shape and properties of the groove on the surface of γ-subunit facilitates transition of the disordered recognition part of the ß-subunit into an α-helix containing approximately the same number of residues in archaea and eukaryotes. In addition, based on the newly obtained data, it was concluded that in archaea and eukaryotes, transition of the γ-subunit to the active state leads to additional contact between the region of switch 1 and C-terminal part of the ß-subunit, which stabilizes helical conformation of the switch.

Structure and Function of Archaeal Translation Initiation Factor 2 Fragments Containing Cys2-Cys2 Motifs.

The heterotrimeric (αßγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (ß and γ) contain the Cys2-Cys2 motif, which is capable of forming a stable zinc finger structure in the presence of zinc ions. In this work, comparative analysis of the fragments containing Cys2-Cys2 motifs in the aIF2ß and aIF2γ structures from different organisms was carried out and their environments in crystals was analyzed. Based on the obtained data, a conclusion was made that the conformation and role of these fragments in the ß- and γ-subunits of the aIF2 are different.

The third structural switch in the archaeal translation initiation factor 2 (aIF2) molecule and its possible role in the initiation of GTP hydrolysis and the removal of aIF2 from the ribosome.

The structure of the γ subunit of archaeal translation initiation factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ-subunit structures in the GTP-bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation initiation factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon-anticodon recognition.

Protein-RNA affinity of ribosomal protein L1 mutants does not correlate with the number of intermolecular interactions.

Ribosomal protein L1, as part of the L1 stalk of the 50S ribosomal subunit, is implicated in directing tRNA movement through the ribosome during translocation. High-resolution crystal structures of four mutants (T217V, T217A, M218L and G219V) of the ribosomal protein L1 from Thermus thermophilus (TthL1) in complex with a specific 80 nt fragment of 23S rRNA and the structures of two of these mutants (T217V and G219V) in the RNA-unbound form are reported in this work. All mutations are located in the highly conserved triad Thr-Met-Gly, which is responsible for about 17% of all protein-RNA hydrogen bonds and 50% of solvent-inaccessible intermolecular hydrogen bonds. In the mutated proteins without bound RNA the RNA-binding regions show substantial conformational changes. On the other hand, in the complexes with RNA the structures of the RNA-binding surfaces in all studied mutants are very similar to the structure of the wild-type protein in complex with RNA. This shows that formation of the RNA complexes restores the distorted surfaces of the mutant proteins to a conformation characteristic of the wild-type protein complex. Domain I of the mutated TthL1 and helix 77 of 23S rRNA form a rigid body identical to that found in the complex of wild-type TthL1 with RNA, suggesting that the observed relative orientation is conserved and is probably important for ribosome function. Analysis of the complex structures and the kinetic data show that the number of intermolecular contacts and hydrogen bonds in the RNA-protein contact area does not correlate with the affinity of the protein for RNA and cannot be used as a measure of affinity.

Disruption of shape complementarity in the ribosomal protein L1-RNA contact region does not hinder specific recognition of the RNA target site.

The formation of a specific and stable complex between two (macro)molecules implies complementary contact surface regions. We used ribosomal protein L1, which specifically binds a target site on 23S rRNA, to study the influence of surface modifications on the protein-RNA affinity. The threonine residue in the universally conserved triad Thr-Met-Gly significant for RNA recognition and binding was substituted by phenylalanine, valine and alanine, respectively. The crystal structure of the mutant Thr217Val of the isolated domain I of L1 from Thermus thermophilus (TthL1) was determined. This structure and that of two other mutants, which had been determined earlier, were analysed and compared with the structure of the wild type L1 proteins. The influence of structural changes in the mutant L1 proteins on their affinity for the specific 23S rRNA fragment was tested by kinetic experiments using surface plasmon resonance (SPR) biosensor analysis. Association rate constants undergo minor changes, whereas dissociation rate constants displayed significantly higher values in comparison with that for the wild type protein. The analysed L1 mutants recognize the specific RNA target site, but the mutant L1-23S rRNA complexes are less stable compared to the wild type complexes.

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.

High-resolution structure of methionine gamma-lyase from Citrobacter freundii.

Pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) is involved in the metabolism of sulfur-containing amino acids. The enzyme is a promising target in some anaerobic pathogens and is effective in cancer-cell treatment. The structure of the MGL holoenzyme from Citrobacter freundii has previously been determined at 1.9 A resolution. By modification of the crystallization procedure, the previously determined structure of C. freundii MGL has been improved to 1.35 A resolution with R and R(free) values of 0.152 and 0.177, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers in detail and to reveal the structurally invariant regions that are responsible for monomer-monomer recognition during the formation of the active enzyme. Details of the mode of cofactor binding and of the flexible regions that may be involved in substrate recognition and binding are also described.

Domain I of ribosomal protein L1 is sufficient for specific RNA binding.

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.

New insights into the interaction of ribosomal protein L1 with RNA.

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.

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.

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 A 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.

Structure of ribosomal protein L1 from Methanococcus thermolithotrophicus. Functionally important structural invariants on the L1 surface.

The crystal structure of ribosomal protein L1 from the archaeon Methanococcus thermolithotrophicus has been determined at 2.7 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 70.1, c = 106.3 A and two molecules per asymmetric unit. The structure was solved by the molecular-replacement method with AMoRe and refined with CNS to an R value of 18.9% and an R(free) of 25.4% in the resolution range 30-2.7 A. Comparison of this structure with those obtained previously for two L1 proteins from other sources (the bacterium Thermus thermophilus and the archaeon M. jannaschii) as well as detailed analysis of intermolecular contacts in the corresponding L1 crystals reveal structural invariants on the molecular surface which are probably important for binding the 23S ribosomal RNA and protein function within the ribosome.

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.