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• W2053361980 abstract "Nuclear pre-mRNA editing by selective adenosine deamination (A-to-I editing) occurs in all organisms from C. elegans to humans. The nucleotide inosine (I) has been observed in viral transcripts and in eukaryotic mRNAs. Inosine derives the deamination of adenosine (A), a process termed RNA editing. Inosine is not only present in mRNA, but also in tRNA, and was first identified in tRNA from yeast.1 In the eukaryotes, seven to eight tRNAs contain I at position 34, whereas in prokaryotes and plant chloroplasts only the tRNAArg2 contains this modification. RNA editing of pre-mRNA is catalyzed by adenosine deaminase acting on RNA (ADARs). The ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify information encoded in the genome, for example, by altering codons in mRNAs.2-4 The functions of ADARs in known substrates suggest that the enzymes function to fine-tune and optimize many biological pathways. Thus, RNA editing alters codon specificity and therefore the amino acid sequence of the encoded protein, resulting in multiple protein products with differing biological functions from a single mRNA precursor. RNA editing represents an important programming mechanism for increasing genetic diversity in eukaryotes. In all cases, pre-mRNA editing requires a double-stranded RNA (dsRNA) structure that is formed between exonic sequences encompassing the editing site and downstream intronic sequences. The genomes of Saccharomyces cerevisiae and prokaryotes do not encode classical ADAR proteins. On the basis of sequence homology to ADARs, a yeast protein has been identified that contains a deaminase domain, but lacks a known RNA-binding motif.5 The deaminase catalyzes deamination of adenosine at position 37 in yeast tRNAala and was, therefore, named adenosine deaminase acting on tRNA 1 and its gene tRNA-specific adenosine deaminase 1 (scADAT1/TAD1).5 ADAT1 proteins have also been cloned from the human,6 mouse,3 and D. melanogaster,7 and the first prokaryotic tRNA editing enzyme from a bacterium (E. coli, TadA) identified.8 Recently, the crystal structures of TadA proteins from three different bacterial species, and in complex form with RNA, have been determined.9-11 By this report, we describe the crystal structure of a tRNA-specific adenosine deaminase (TAD) from Streptococcus pyogenes, which was determined at 2.0 Å resolution by multiwavelength anomalous dispersion (MAD) using bound zinc ion. The structure reveals an enzyme that form a homodimer with each monomer consisting of typical α/β fold domains and a long α-helical of C-terminus, a unique structure that has been observed in other Tad families. The zinc ion is tetrahedral coordinated by highly conserved residues, one His, two Cysteines, and one water molecule. Our structural results provide insight into the function of conserved residues within the TAD superfamily. The cloning, purification, and crystallization of TadA from Streptococcus pyogenes has been reported.12 Briefly, His-tagged TadA in pET22b vector was expressed in an Escherichia coli strain BL21 (DE3). The Zn-MAD data sets were collected using a synchrotron radiation source at beamline 4A of Pohang Light Source (PLS, South Korea). MAD data were collected at three wavelengths (peak = 1.2834, edge = 1.2827, remote = 1.2573). The crystals belonged to the space group P42212 with a = b = 80.091 Å, c = 81.104 Å. Data were processed within the program HKL2000 and SCALEPACK.13 The structure of TadA from S. pyogenes was determined by the multiwavelength anomalous diffraction method using zinc ion of crystal. The phases were calculated with the SOLVE and RESOLVE14 programs with a mean figure of merit of 0.60 at 2.8 Å resolution. The MAD phasing statistics and refinement statistics are presented in Table I. The model was completed by iterative cycles of model building with Coot15 and refinement with CNS16 yielding a final model containing 173 amino acids, one Zn(II) ion, one phosphate ion, and 147 water molecules for TadA. The metal content of the crystal was assessed by X-ray fluorescence experiments carried out at Pohang Light Source Beamline 4A near the κ edge for zinc (1.38 Å). We carried out the structure analyses using the following computer programs: Swiss-Pdb-Viewer and Coot for superposition of molecules and secondary structure-based alignment; SSAP server17 for calculation of the root-mean-square deviations (RMSDs); ES-Pript18 for preparation of the secondary structure alignment figure; Pymol19 for the depiction of structure; and GRASP20 for the calculation and depiction of electrostatic potentials at the molecular surface. Other orthologs used in the structural comparison were Staphylococcus aureus (PDB code 2B3J),9 E. coli (PDB code 1Z3A),10 A. aeolicus (PDB code 1WWR).11 The structure of TadA from Streptococcus pyogenes (spTadA) was determined to a resolution of 2.0 Å using the multiwavelength anomalous dispersion (MAD) method with zinc ion. The crystal shows symmetry of the space group P42212 and contains one molecule in the asymmetric unit. The monomer is a classical α/β/α protein of approximate dimensions 31 × 33 × 58 Å3 [Fig. 1(a)]. The structure consists of a central β-sheet with two α-helices (α1, α6) on one side of the sheet and three α-helices (α3–α5) on the other side. The overall folding of spTadA is similar to that of other bacteria, TadA from Staphylococcus aureus (PDB code 2B3J),9 Escherichia coli (PDB code 1Z3A),10 Aquilfex aeolicus (PDB code 1WWR),11 with an overall Cα rms deviations of 0.75, 0.75, and 1.45 Å, respectively. The main differences are found in the loop and 310-helix (α5) between β4 and β5, the additional region of the N-terminal and the long C-terminal α-helix. A superposed illustration is shown in Figure 1(b). Additional structural analyses show that spTadA have high similarity in the shape of the active site cavities and distribution of conserved residues on and around the surface of the cavities. Although the residues that comprised the cavities are mostly conserved, there are still some nonconserved residues distributed on the surface of the cavities. The notable C-terminal long helices functioning to cap the active site of the cavity in spTadA structures may play role in binding specific tRNA substrates. The simulated annealing omit map showed a marked electron density peak located within 2.0–2.5 Å from His65, Cys95, and Cys98. The Zn2+ absorption edge was determined to be 1.38 Å in an X-ray absorption experiment. The position of the bound Zn2+ in spTadA is very similar to that in the other TadA structures (rms deviation 0.2 Å). The Zn2+ ion located at the bottom of the active site is coordinated by the Nδ1 atom of His65 (2.1 Å), Sγ atom of Cys95 (2.4 Å), Sγ atom of Cys98 (2.4 Å), and a water molecule (2.46 Å) with a slightly distorted tetragonal geometry. Interestingly, we found a phosphate ion near the active site that is formed by the hydrogen bonding with Nϵ2 of His65 (2.7 Å). When our structure was superimposed with the complex-tad structure (pdb1WWR), this position coincided with the RNA substrate [Fig. 1(c)]. (A) Overall structure A schematic ribbon diagram of the overall structure of the tRNA-specific adenosine deaminase (residues 5-173) from S. pyogenes (spTad A). The N- and C-termini of the protein are labeled, and the helices and sheets are numbered. Zn (II) and the phosphate ion are shown in gray sphere and ball-and-stick models, respectively. (B) Superimposition of spTadA and other proteins. The overall structure of spTadA is superimposed on other TadA proteins in a ribbon model representation. The blue, yellow, red and green colors are shown to S. pyogenes TadA, A. aeolicus TadA (1WWR), E. coli TadA (1Z3A), and Staphylococcus aureus TadA (2B3J), respectively. The red circles are shown in the main differences. (C) Comparison of active sites between spTadA and saTadA-complexed with RNA. A detailed depiction of the interacting residues of the active site compared to Staphylococcus aureus Tad A (saTadA) is shown by ball-and-stick models. In saTadA, the His residue has the π–π interaction with PURINE RIBOSIDE-5′-MONOPHOSPHATE (C10 H13 N4 O7 P) (PR5). Green residues are spTadA, yellow residues the saTad A (2B3J), blue the phosphate ion, PO4− (spTad), and red is PR5 (2B3J). (D) The dimer interface of spTadA. The monomers interact with each other symmetrically. The residues participated in forming the interfaces are shown as ball-and-stick models and the interaction with another monomer. The hydrogen bonding and hydrophobic interactions are major forces in forming the dimer interface. The purified spTadA ran at an apparent molecular weight of 38 kDa on a gel-filtration column, approximately twice the expected molecular weight of 19 kDa. The dimer appears to be the physiological condition with the crystallographic packing showing that the spTadA forms a dimer when in a solution. The dimer interface is formed around the helix α4 with a crystallographic twofold symmetry through hydrogen bonding, salt bridging, and hydrophobic interactions [Fig. 1(d)]. Dimer formation decreases the accessible surface by 1,684.3 Å2 per monomer or 15.4% of the total surface of the monomer. Among the buried surface residues, 48.4% are hydrophobic, 19.4% are polar, and 29.0% are charged indicating that the majority of the dimeric interface is formed by hydrophobic and nonpolar interactions. From the structural aspects, dimerization appears to be important for specific binding with tRNA and enzyme functions. Firstly, the shape and charge distribution of the dimer surface is suitable to bind with tRNA and secondly, most of the dimer interface is nonpolar. The formation of a dimer, therefore, would be energetically favorable compared with the monomer in solution. In summary, we have determined the three-dimensional structure of the spTadA at 2.0 Å. The arrangements of the active site residues and the interactions among them have been elucidated and provide a clearer understanding of the mechanism for catalysis by the enzyme. Structure comparisons reveal some different structural characteristics between spTadA and other TadAs species. We thank Dr. H. S. Lee and his staff for assistance during data collection at beamline 4A of Pohang Light Source, Korea." @default.
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• W2053361980 date "2007-06-06" @default.
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• W2053361980 title "Crystal structure of the tRNA-specific adenosine deaminase from Streptococcus pyogenes" @default.
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• W2053361980 doi "https://doi.org/10.1002/prot.21456" @default.
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