Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

Structural model for the selenocysteine-specific elongation factor SelB

A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria. The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu. The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP. In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB. Information on the formation of the selenocysteyl-binding pocket is presented. A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors.     

Structure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson-Crick base-pairs apart from the GpU are essential for interaction with SelB.     

Structural basis of mRNA cap recognition by proteins

Crystal structures have recently become available for two proteins (VP39 and eIF4E) complexed with their cognate ligand - the mRNA cap. Despite their total structural dissimilarity, both proteins bind N7-methylguanine between two parallel aromatic sidechains. The resulting stacked arrangement governs their high specificity for the alkylated form of the nucleobase.     

Structural features in aminoacyl-tRNAs required for recognition by elongation factor Tu

In bacterial polypeptide synthesis aminoacyl-tRNA (aa-tRNA) bound to elongation factor Tu (EF-Tu) and GTP is part of a crucial intermediate ribonucleoprotein complex involved in the decoding of messenger RNA. The conformation and topology as well as the affinity of the macromolecules in this ternary aa-tRNA X EF-Tu X GTP complex are of fundamental importance for the nature of the interaction of the complex with the ribosome. The structural elements of aa-tRNA required for interaction with EF-Tu and GTP and the resulting functional implications are presented here.     

Purification and characterization of hexahistidine-tagged elongation factor SelB

The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNASec. In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion. In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented. After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet. Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNASec. In addition, the fusion protein is fully functional in mediating UGA read-through in vivo. It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation.     

Structural insight into a molecular switch in tandem winged-helix motifs from elongation factor SelB

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3-A resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6-A resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.     

Structural basis of coagulation factor V recognition for cleavage by RVV-V

Russell's viper venom factor V (FV) activator (RVV-V) is a thrombin-like proteinase that specifically cleaves the Arg1545-Ser1546 bond of FV. Here we present the crystal structure of RVV-V in complex with the FV14 peptide (residues 1533-1546 of human FV) determined at 1.8? resolution. The structure reveals multiple interactions between RVV-V and the seven residues, Ile1539 (P(7))-Arg1545 (P(1)), of the cleaved substrate. Comparison with substrate-free structures reveals conformational changes of the RVV-V loops upon substrate binding, suggesting that the multiple interactions are mediated by an induced-fit mechanism. The results provide an explanation for the narrow specificity of RVV-V.     

Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

Selenocysteine (Sec), the 21^st amino acid in translation, uses its specific tRNA (tRNA^Sec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA^Sec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA^Sec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA^Sec allows SelB to specifically recognize tRNA^Sec and characteristically place it at the ribosomal A-site.     

Structural basis for ligand recognition by integrins

Integrins, the major cell surface receptors mediating cell-extracellular matrix (ECM) adhesion, are central to the basic physiology underlying all multicellular organisms. As the complexity of animal body architecture increased, integrins were forced to acquire recognition capabilities toward the wide variety of ECM ligands and cell surface counter-receptors that emerged during evolution. Structural determination of the integrin-ligand complexes for both I domain-containing and non-I domain-containing integrins revealed two fundamentally different types of integrin-binding surfaces. In addition, recent advances in the biochemical and pharmacological characterization of the integrin-ligand interactions are beginning to reveal how integrins achieve specific recognition of wide variety of ligands using a small binding cleft at the subunit interface common to all integrins.     

Selenocysteine tRNA-specific elongation factor SelB is a structural chimaera of elongation and initiation factors

In all three kingdoms of life, SelB is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a UGA stop codon in the presence of a downstream mRNA hairpin loop. Here, we present the X-ray structures of SelB from the archaeon Methanococcus maripaludis in the apo-, GDP- and GppNHp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. All three SelB structures reveal an EF-Tu:GTP-like domain arrangement. Upon binding of the GTP analogue GppNHp, a conformational change of the Switch 2 region in the GTPase domain leads to the exposure of SelB residues involved in clamping the 5' phosphate of the tRNA. A conserved extended loop in domain III of SelB may be responsible for specific interactions with tRNA(Sec) and act as a ruler for measuring the extra long acceptor arm. Domain IV of SelB adopts a beta barrel fold and is flexibly tethered to domain III. The overall domain arrangement of SelB resembles a 'chalice' observed so far only for initiation factor IF2/eIF5B. In our model of SelB bound to the ribosome, domain IV points towards the 3' mRNA entrance cleft ready to interact with the downstream secondary structure element.     

Structural basis for recognition of S-adenosylhomocysteine by riboswitches

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.     

Structural basis for anticodon recognition by methionyl-tRNA synthetase

In the 2.7-A resolution crystal structure of methionyl-tRNA synthetase (MetRS) in complex with tRNA(Met) and a methionyl-adenylate analog, the tRNA anticodon loop is distorted to form a triple-base stack comprising C34, A35 and A38. A tryptophan residue stacks on C34 to extend the triple-base stack. In addition, C34 forms Watson-Crick-type hydrogen bonds with Arg357. This structure resolves the longstanding question of how MetRS specifically recognizes tRNA(Met).     

Structural basis for NHERF recognition by ERM proteins

The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic alpha helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.     

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor I(m) (CF I(m)), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I(m), comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I(m)-RNA complex. These structures show that two CF I(m)68RRM molecules bind to the CF I(m)25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF I(m)25-CF I(m)68RRM heterotetramer, providing structural basis for the mechanism by which CF I(m) binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF I(m)68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF I(m)68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF I(m)68 makes an essential contribution to pre-mRNA binding.     

Crystal structure of an mRNA-binding fragment of Moorella thermoacetica elongation factor SelB

SelB is an elongation factor needed for the co-translational incorporation of selenocysteine. Selenocysteine is coded by a UGA stop codon in combination with a specific downstream mRNA hairpin. In bacteria, the C-terminal part of SelB recognizes this hairpin, while the N-terminal part binds GTP and tRNA in analogy with elongation factor Tu (EF-Tu). We present the crystal structure of a C-terminal fragment of SelB (SelB-C) from Moorella thermoacetica at 2.12 A resolution, solved by a combination of selenium and yttrium multiwavelength anomalous dispersion. This 264 amino acid fragment contains the entire C-terminal extension beginning after the EF-Tu-homologous domains. SelB-C consists of four similar winged-helix domains arranged into the shape of an L. This is the first example of winged-helix domains involved in RNA binding. The location of conserved basic amino acids, together with data from the literature, define the position of the mRNA-binding site. Steric requirements indicate that a conformational change may occur upon ribosome interaction. Structural observations and data in the literature suggest that this change happens upon mRNA binding.     

Structural basis for glycogen recognition by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.