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.     

Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (αβ)₂ family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 Å containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Å. The functional enzyme resembles previously known α₂ and α₄ endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.     

Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.     

Crystal structure of human selenocysteine tRNA

Selenocysteine (Sec) is the 21st amino acid in translation. Sec tRNA (tRNA^Sec) has an anticodon complementary to the UGA codon. We solved the crystal structure of human tRNA^Sec. tRNA^Sec has a 9-bp acceptor stem and a 4-bp T stem, in contrast with the 7-bp acceptor stem and the 5-bp T stem in the canonical tRNAs. The acceptor stem is kinked between the U6:U67 and G7:C66 base pairs, leading to a bent acceptor-T stem helix. tRNA^Sec has a 6-bp D stem and a 4-nt D loop. The long D stem includes unique A14:U21 and G15:C20a pairs. The D-loop:T-loop interactions include the base pairs G18:U55 and U16:U59, and a unique base triple, U20:G19:C56. The extra arm comprises of a 6-bp stem and a 4-nt loop. Remarkably, the D stem and the extra arm do not form tertiary interactions in tRNA^Sec. Instead, tRNA^Sec has an open cavity, in place of the tertiary core of a canonical tRNA. The linker residues, A8 and U9, connecting the acceptor and D stems, are not involved in tertiary base pairing. Instead, U9 is stacked on the first base pair of the extra arm. These features might allow tRNA^Sec to be the target of the Sec synthesis/incorporation machineries.     

Crystal structure and functional analysis of lipoamide dehydrogenase from Mycobacterium tuberculosis

We report the 2.4 A crystal structure for lipoamide dehydrogenase encoded by lpdC from Mycobacterium tuberculosis. Based on the Lpd structure and sequence alignment between bacterial and eukaryotic Lpd sequences, we generated single point mutations in Lpd and assayed the resulting proteins for their ability to catalyze lipoamide reduction/oxidation alone and in complex with other proteins that participate in pyruvate dehydrogenase and peroxidase activities. The results suggest that amino acid residues conserved in mycobacterial species but not conserved in eukaryotic Lpd family members modulate either or both activities and include Arg-93, His-98, Lys-103, and His-386. In addition, Arg-93 and His-386 are involved in forming both "open" and "closed" active site conformations, suggesting that these residues play a role in dynamically regulating Lpd function. Taken together, these data suggest protein surfaces that should be considered while developing strategies for inhibiting this enzyme.     

Crystal structure and functional analysis of mycobacterial erythromycin resistance methyltransferase Erm38 reveals its RNA-binding site

Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from Mycobacterium smegmatis in apoprotein and cofactor-bound forms. Based on structural analysis and mutagenesis, we identified several catalytically critical, positively charged residues at a putative RNA-binding site. We found that mutation of any of these sites is sufficient to abolish methylation activity, whereas the corresponding RNA-binding affinity of Erm38 remains unchanged. The methylation reaction thus appears to require a precise ensemble of amino acids to accurately position the RNA substrate, such that the target nucleotide can be methylated. In addition, we computationally constructed a model of Erm38 in complex with a 32-mer RNA substrate. This model shows the RNA substrate stably bound to Erm38 by a patch of positively charged residues. Furthermore, a π-π stacking interaction between a key aromatic residue of Erm38 and a target adenine of the RNA substrate forms a critical interaction needed for methylation. Taken together, these data provide valuable insights into Erm–RNA interactions, which will aid subsequent structure-based drug design efforts.     

Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.     

Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe

Decapping is a key step in both general and nonsense-mediated 5' --> 3' mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of a C-terminally truncated Schizosaccharomyces pombe Dcp2p reveals two distinct domains: an all-helical N-terminal domain and a C-terminal domain that is a classic Nudix fold. The C-terminal domain of both Saccharomyces cerevisiae and S. pombe Dcp2p proteins is sufficient for decapping activity, although the N-terminal domain can affect the efficiency of Dcp2p function. The binding of Dcp2p to Dcp1p is mediated by a conserved surface on its N-terminal domain, and the N-terminal domain is required for Dcp1p to stimulate Dcp2p activity. The flexible nature of the N-terminal domain relative to the C-terminal domain suggests that Dcp1p binding to Dcp2p may regulate Dcp2p activity through conformational changes of the two domains.     

Crystal structure of ISG54 reveals a novel RNA binding structure and potential functional mechanisms

Interferon-stimulated gene 56 (ISG56) family members play important roles in blocking viral replication and regulating cellular functions, however, their underlying molecular mechanisms are largely unclear. Here, we present the crystal structure of ISG54, an ISG56 family protein with a novel RNA-binding structure. The structure shows that ISG54 monomers have 9 tetratricopeptide repeat-like motifs and associate to form domain-swapped dimers. The C-terminal part folds into a super-helical structure and has an extensively positively-charged nucleotide-binding channel on its inner surface. EMSA results show that ISG54 binds specifically to some RNAs, such as adenylate uridylate (AU)-rich RNAs, with or without 5′ triphosphorylation. Mutagenesis and functional studies show that this RNA-binding ability is important to its antiviral activity. Our results suggest a new mechanism underlying the antiviral activity of this interferon-inducible gene 56 family member.     

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNA^Cys (Sep-tRNA^Cys) into Cys-tRNA^Cys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) to selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNA^Cys and tRNA^Sec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNA^Sec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNA^Sec to Cys-tRNA^Sec, and that Sep is crucial for SepCysS recognition.     

Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.     

Crystal structure of tRNA adenosine deaminase (TadA) from Aquifex aeolicus

The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNA(Arg-2). This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-A resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an alpha/beta/alpha three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the beta4 and beta5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop.     

Genome-wide analysis reveals the functional and expressional correlation between RhoGAP and RhoGEF in mouse

Rho GTPases play essential roles in various life activities. Rho GTPase-activating protein (RhoGAP) and Rho guanine nucleotide exchange factor (RhoGEF) are the main regulators of Rho GTPases. RhoGAP, RhoGEF and Rho make up a molecular switch and exert crucial roles in signaling pathways. The genome-wide studies can provide us a comprehensive information of special protein family, but the genome-wide information of RhoGAP and RhoGEF families are still lacking in the mammal lineage. Here, we report the correlations between mouse RhoGAPs and RhoGEFs in gene quantities, evolution, molecular function, and their expression levels in heart embryonic development and cardiovascular medicine treatment at genome-wide scale. Besides, we find that the 3D structures of RhoGAP domains between different species are highly conserved, but that of RhoGEF domains are variable between species. Our present study contributes to a better understanding of the complex regulation mechanisms of RhoGAP and RhoGEF families.     

Crystal structure of the tRNA 3' processing endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.     

Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.     

Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding

Staphylococci use cell wall‐anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall‐anchored protein, uro‐adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain‐truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.     

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.