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.

Water clusters in the nucleotide-binding pocket of the protein aIF2γ from the archaeon Sulfolobus solfataricus: Proton transmission.

In Archaea and Eukaryotes, the binding of Met-tRNAi(Met) to the P-site of the ribosome is mediated by translation initiation factor 2 (a/eIF2) which consists of three subunits: α, ß and γ. Here, we present the high-resolution structure of intact aIF2γ from Sulfolobus solfataricus (SsoIF2γ) in complex with GTP analog, GDPCP. The comparison of the nucleotide-binding pockets in this structure and in the structure of the ribosome-bound form of EF-Tu reveals their close conformation similarity. The nucleotide-binding pocket conformation observed in this structure could be consider as corresponding to intermediate conformation of EF-Tu nucleotide-binding pocket in its transition from the GTP-bound form to the GDP-bound one. Three clusters of well defined water molecules are associated with amino acid residues of the SsoIF2γ nucleotide-binding pocket and stabilize its conformation. We suppose that two water bridges between the oxygen atoms of the GTP γ-phosphate and negatively charged residues of the pocket can serve as ways to transmit protons arising from the catalytic reaction.

Binding of the 5'-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus.

The heterotrimeric archaeal IF2 orthologue of eukaryotic translation initiation factor 2 consists of the α-subunit, ß-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs. The biological implication of these findings is being discussed.

The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

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.

Conformational transitions in the γ subunit of the archaeal translation initiation factor 2.

In eukaryotes and archaea, the heterotrimeric translation initiation factor 2 (e/aIF2) is pivotal for the delivery of methionylated initiator tRNA (Met-tRNA(i)) to the ribosome. It acts as a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states. Recent studies show that eIF2 can also exist in a long-lived eIF2γ-GDP-P(i) (inorganic phosphate) active state. Here, four high-resolution crystal structures of aIF2γ from Sulfolobus solfataricus are reported: aIF2γ-GDPCP (a nonhydrolyzable GTP analogue), aIF2γ-GDP-formate (in which a formate ion possibly mimics P(i)), aIF2γ-GDP and nucleotide-free aIF2γ. The structures describe the different states of aIF2γ and demonstrate the conformational transitions that take place in the aIF2γ `life cycle'.

Revisiting the Haloarcula marismortui 50S ribosomal subunit model.

The structure of the large ribosomal subunit from the halophilic archaeon Haloarcula marismortui (Hma) is the only crystal structure of an archaeal ribosomal particle that has been determined to date. However, the first model of the Hma 50S ribosomal subunit contained some gaps: the structures of functionally important mobile lateral protuberances were not visualized. Subsequently, some parts of the P (L12) stalk base were visualized at 3.0 Å resolution [Kavran & Steitz (2007), J. Mol. Biol. 371, 1047-1059]: the RNA-binding domain of r-protein P0 (L10), the C-terminal domain of L11 and helices 43 and 44 of the 23 S rRNA. Here, the 2.4 Å resolution electron-density map of the Hma 50S ribosomal subunit was revisited and approximately two-thirds of the P0 protein, residues 1-58 of the N-terminal domains of two P1 protein molecules, residues 130-156 of L11, the full-length r-protein LX, nucleotides 2137-2149 and 2226-2237 of the 23S rRNA helix H76 forming the L1 stalk, nucleotides 2339-2343 of the 23S rRNA (contacting L5 protein) and loops 29-34 and 108-128 of protein L5 could be visualized. Thus, this paper provides a supplemented version of the Hma 50S ribosomal subunit model.

Crystal structure of the archaeal translation initiation factor 2 in complex with a GTP analogue and Met-tRNAf(Met.).

Heterotrimeric aIF2αßγ (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.

High-resolution crystal structure of spectrin SH3 domain fused with a proline-rich peptide.

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the two-fold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.

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.

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.

Structure of a two-domain N-terminal fragment of ribosomal protein L10 from Methanococcus jannaschii reveals a specific piece of the archaeal ribosomal stalk.

Ribosomal stalk is involved in the formation of the so-called "GTPase-associated site" and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 A resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.

Structural and thermodynamic studies of Bergerac-SH3 chimeras.

Bergerac-type chimeras of spectrin SH3 were designed by extending a beta-hairpin by eight amino acids so that the extension protruded from the domain body like a "nose" being exposed to the solvent. A calorimetric study of several Bergerac-SH3 variants was carried out over a wide range of pH values and protein concentrations and the three-dimensional structure of one of them, SHH, was determined. X-ray studies confirmed that the nose had a well defined beta-structure whilst the chimera formed a stable tetramer within the crystal unit because of four tightly packed noses. In the pH range of 4-7 the heat-induced unfolding of some chimeras was complex and concentration dependent, whilst at pH values below 3.5, low protein concentrations of all the chimeras studied, including SHH, seemed to obey a monomolecular two-state unfolding model. The best set of data was obtained for the SHA variant, the unfolding heat effects of which were systematically higher than those of the WT protein (about 16.4 kJ/mol at 323 K), which may be close to the upper limit of the enthalpy gain due to 10 residue beta-hairpin folding. At the same time, the chimeras with high nose stability, which, like SHH, have a hydrophobic (IVY) cluster on their surface, showed a lower apparent unfolding heat effect, much closer to that of the WT protein. The possible reasons for this difference are discussed.

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.

Crystal structure of the intact archaeal translation initiation factor 2 demonstrates very high conformational flexibility in the alpha- and beta-subunits.

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (alpha, beta, and gamma), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-A resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2alphagamma and aIF2betagamma heterodimers revealed high conformational flexibility in the alpha- and beta-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the gamma-subunit, domain 3 of the alpha-subunit, and the N-terminal alpha-helix of the beta-subunit, and two mobile "wings," formed by domains 1 and 2 of the alpha-subunit, the central part, and the zinc-binding domain of the beta-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the gamma-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.

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 interactions of the translation initiation factor 2 from archaea with guanine nucleotides and initiator tRNA.

Heterotrimeric a/eIF2alphabetagamma (archaeal homologue of the eukaryotic translation initiation factor 2 with alpha, beta and gamma subunits) delivers charged initiator tRNA (tRNAi) to the small ribosomal subunit. In this work, we determined the structures of aIF2gamma from the archaeon Sulfolobus solfataricus in the nucleotide-free and GDP-bound forms. Comparison of the free, GDP and Gpp(NH)p-Mg2+ forms of aIF2gamma revealed a sequence of conformational changes upon GDP and GTP binding. Our results show that the affinity of GDP to the G domain of the gamma subunit is higher than that of Gpp(NH)p. In analyzing a pyrophosphate molecule binding to domain II of the gamma subunit, we found a cleft that is very suitable for the acceptor stem of tRNA accommodation. It allows the suggestion of an alternative position for Met-tRNA i Met on the alphagamma intersubunit dimer, at variance with a recently published one. In the model reported here, the acceptor stem of the tRNAi is approximately perpendicular to that of tRNA in the ternary complex elongation factor Tu-Gpp(NH)p-tRNA. According to our analysis, the elbow and T stem of Met-tRNA i Met in this position should make extensive contact with the alpha subunit of aIF2. Thus, this model is in good agreement with experimental data showing that the alpha subunit of aIF2 is necessary for the stable interaction of aIF2gamma with Met-tRNA i Met.