FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2074731176> ?p ?o ?g. }

• W2074731176 endingPage "16008" @default.
• W2074731176 startingPage "16002" @default.
• W2074731176 abstract "The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-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-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α 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 β4 and β5 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. The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-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-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α 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 β4 and β5 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. Inosine (Fig. 1A) was found at the first (“wobble”) position of the tRNA anticodon 40 years ago (1Holley R.W. Everett G.A. Madison J.T. Zamir A. J. Biol. Chem. 1965; 240: 2122-2128Abstract Full Text PDF PubMed Google Scholar). Crick postulated (2Crick F.H. J. Mol. Biol. 1966; 19: 548-555Crossref PubMed Scopus (1306) Google Scholar) that inosine is able to pair with U, C, and A. The codons corresponding to each inosine-bearing tRNA are synonymous, which contributes to decreasing the number of isoacceptor tRNAs (3Marck C. Grosjean H. RNA. 2002; 8: 1189-1232Crossref PubMed Scopus (285) Google Scholar). In eukaryotes, the wobble positions of eight cytoplasmic tRNAs (seven in Saccharomyces cerevisiae) bear inosine (4Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (815) Google Scholar), which is generated by the posttranscriptional hydrolytic deamination of adenosine (5Auxilien S. Crain P.F. Trewyn R.W. Grosjean H. J. Mol. Biol. 1996; 262: 437-458Crossref PubMed Scopus (92) Google Scholar). In most bacteria and plant chloroplasts, only tRNAArg-2 (Fig. 1B) has the inosine modification (4Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (815) Google Scholar). The enzymes that catalyze inosine generation were cloned recently (6Gerber A.P. Keller W. Trends Biochem. Sci. 2001; 26: 376-384Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar). In eukaryotes, a heterodimeric enzyme composed of two sequence-related subunits (Tad2p/ADAT2 1The abbreviations used are: ADAT, adenosine deaminase acting on tRNA; TadA, tRNA adenosine deaminase; CDA, cytidine deaminase; CD, cytosine deaminase; GD, guanine deaminase; MES, 4-morpholinoethanesulfonic acid. and Tad3p/ADAT3) is responsible for this modification (6Gerber A.P. Keller W. Trends Biochem. Sci. 2001; 26: 376-384Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In most bacteria and plant chloroplasts, a tRNA adenosine deaminase (TadA) edits the adenosine residue at the wobble position of tRNAArg-2 (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar). TadA shares homology with Tad2p and is considered to form a homodimer. TadA contains the consensus motif (C/H)XEXnPCXXCofthe cytidine deaminase (CDA) superfamily, which includes diverse deaminases acting on bases, nucleosides, nucleotides, and nucleic acids (8Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar, 9Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar, 13Teng B. Burant C.F. Davidson N.O. Science. 1993; 260: 1816-1819Crossref PubMed Scopus (496) Google Scholar, 14Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 15Muramatsu M. Sankaranand V.S. Anant S. Sugai M. Kinoshita K. Davidson N.O. Honjo T. J. Biol. Chem. 1999; 274: 18470-18476Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, 16Jarmuz A. Chester A. Bayliss J. Gisbourne J. Dunham I. Scott J. Navaratnam N. Genomics. 2002; 79: 285-296Crossref PubMed Scopus (590) Google Scholar). This consensus motif forms the core of the active site and chelates a zinc ion tetrahedrally, and a common zinc-assisted deamination mechanism has been proposed (8Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar, 9Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar). In contrast, the mechanisms of substrate recognitions are diverse. Based on the complex structures solved thus far, the recognition mechanisms have been reported for cytidine (8Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar, 9Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar), cytosine (10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and guanine (11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The only solved structure of a deaminase acting on polymeric nucleic acids is that of yeast CDD1 (12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar), which edits both mRNA and cytidine (17Dance G.S. Beemiller P. Yang Y. Mater D.V. Mian I.S. Smith H.C. Nucleic Acids Res. 2001; 29: 1772-1780Crossref PubMed Scopus (29) Google Scholar). Based on the available structures, molecular models of other deaminases acting on nucleic acids has been made and their substrate recognition modes have been reported (12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar, 18Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (126) Google Scholar). In this study, we solved the crystal structure of TadA from Aquifex aeolicus at a 1.8-Å resolution. This is the first structure of a deaminase acting on tRNA. TadA forms a dimer, whereas the yeast CDD1 forms a tetramer. We made a docking model of TadA with tRNA. Protein Preparation—The A. aeolicus tadA gene was PCR-amplified from the genomic DNA and subcloned into pET28b (Novagen) between the NdeI and SalI sites. The recombinant protein consists of the 151 amino acid residues from A. aeolicus TadA and 20 additional vector-encoded His tag residues (MGSSHHHHHHSSGLVPRGSH) at the N terminus for affinity purification. Escherichia coli strain BL21(DE3) CodonPlus (Stratagene) was transformed with the plasmid. For protein overexpression, the cells were grown in LB medium at 37 °C to an A600 of 0.6 and then the expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 3 h. The cells were harvested and sonicated in 20 mm Tris-HCl buffer (pH 8.5) containing 500 mm NaCl, 10 mm imidazole, 1.4 mm 2-mercaptoethanol, and a protease inhibitor mixture, Complete EDTA-free (Roche Applied Science). The insoluble cell debris was removed by centrifugation at 15,000 × g for 10 min at 4 °C. The supernatant was heat-treated at 72 °C for 20 min to denature the E. coli proteins. The heat-treated mixture was centrifuged at 15,000 × g for 15 min at 4 °C. The supernatant was applied to a 10-ml column of nickel-nitrilotriacetic acid Superflow (Qiagen) equilibrated with 20 mm Tris-HCl buffer (pH 8.5) containing 500 mm NaCl, 10 mm imidazole, and 1.4 mm 2-mercaptoethanol. The protein was eluted in one step with 20 mm HEPES-NaOH buffer (pH 7.5) containing 300 mm NaCl, 250 mm imidazole, and 1.4 mm 2-mercaptoethanol. Four volumes of 20 mm HEPES-NaOH buffer (pH 7.5) containing 1.4 mm 2-mercaptoethanol were added to the eluate to reduce the NaCl concentration to less than 100 mm, and then this solution was loaded onto a UnoS column (Bio-Rad) using an ÄKTA system (Amersham Biosciences). The protein was eluted with a linear gradient of 0.1–0.5 m NaCl in 20 mm HEPES-NaOH buffer (pH 7.5) containing 1.4 mm 2-mercaptoethanol. Two peaks appeared in the chromatogram. Each peak fraction was individually diluted and loaded onto the UnoS column again. Both elution patterns were the same as the first elution. Two peaks appeared at the corresponding salt concentrations. The two-peak fractions were mixed and dialyzed against 10 mm HEPES-NaOH buffer (pH 7.5) containing 400 mm NaCl and 5 mm 2-mercaptoethanol. The final purity of the protein was > 95% as monitored by SDS-PAGE, and 1 mg of protein was obtained per liter of LB medium. Crystallization and Data Collection—Prior to crystallization, the protein was concentrated to 8.4 mg/ml by ultrafiltration. For crystallization, the hanging drop vapor diffusion method was used by mixing 1 μl of the protein solution with 1 μl of 50 mm MES-NaOH buffer (pH 5.6) containing 200 mm KCl, 10 mm MgCl2, and 5% polyethylene glycol 8000 and equilibrating the mixture against 500 μl of the reservoir solution composed of 45 mm MES-NaOH buffer (pH 5.6) containing 400 mm NaCl, 180 mm KCl, 9 mm MgCl2, and 4.5% polyethylene glycol 8000 at 20 °C. Crystals were grown in 3 days to dimensions of ∼0.5 × 0.3 × 0.15 mm3. The diffraction data set of the crystal was collected at beamline BL26B1 at SPring-8 (Harima, Japan) to a 1.8-Å resolution. Before flash-cooling, the crystal was transferred into a cryoprotective solution composed of 55 mm MES-NaOH buffer (pH 5.6) containing 35% (v/v) glycerol, 220 mm KCl, 11 mm MgCl2, and 5.5% polyethylene glycol 8000. The data were processed and reduced using the HKL2000 program (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The crystal (space group P21; unit-cell parameters, a = 43.2 Å, b = 152.0 Å, c = 54.1 Å, β = 113.4°) contains four molecules per asymmetric unit with a solvent content of 41%. Phase Determination and Structure Refinement—We solved the structure by the molecular replacement method. The initial model was the N-terminal 94 amino acid residues of yeast cytosine deaminase (CD) (Protein Data Bank code 1UAQ), and its sequence was replaced by the sequence of A. aeolicus TadA by the 3D-PSSM server. The N-terminal amino acid residues (Met15-Val108 of yeast CD and Leu7-Ile98 of A. aeolicus TadA) share 31% sequence identity (Fig. 2). We carried out molecular replacement using the program MOLREP (20Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar, 21Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Three monomers were found and were first subjected to rigid body refinement using the data set up to a 2.5-Å resolution. After several rounds of Cartesian coordinate energy minimization, simulated annealing, and B factor refinement using the program CNS (22Adams P.D. Pannu N.S. Read R.J. Brunger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar) as well as manual revision and building of the model using the program O (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), R and Rfree decreased to 34.6 and 36.7%, respectively. Two of the three monomers formed a dimer, so we used this dimer as a search model and repeated the molecular replacement. Two dimers then were found, and the structure refinement was performed in the same way using the data set from a 50.0–1.8-Å resolution. The model refinement finally converged to an R value of 19.8% and an Rfree value of 24.8% with good stereochemistry. A Ramachandran plot analysis using the program PROCHECK (24Laskowski R.A. MacArthor M.W. Moris A.L. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) showed that 89.8% of the residues are in the most favored regions and 10.2% are in the additionally allowed regions. Data collection and model-refinement statistics are shown in Table I. The electron density of the main chain was clear for all of the TadA residues with the exception of the partially disordered residues Pro119-Asn122 and the two C-terminal residues. In addition, 3, 8, and 9 of the 20 vector-encoded His tag residues were visible at the N terminus in molecules A, B, and C, respectively.Table IData collection and model-refinement statisticsData collectionSpace groupP21Unit-cell parametersa = 43.2 Åb = 152.0 Åc = 54.1 Åβ = 113.4°Resolution range (Å)50.0-1.8Unique reflections56,975Redundancy (last shell)3.1(1.7)Completeness (%) (last shell)96.5(77.7)I/σ (last shell)19.1(2.0)Rsym (%) (last shell)aRsym = Σhkl Σj|Ij(hkl) - 〈Ij(hkl)〉|/Σhkl ΣjI(hkl), where Ij(hkl) and 〈Ij(hkl)〉 are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.7.7(17.6)Structure RefinementNo. of reflections: working set/test set54,092/2,883No. of protein atoms4,972No. of water molecules520No. of ion atoms4Rfactor (%): working set/test setbRfactor = Σ|Fobs - kFcalc|/ΣhklFobs, where k is a scale factor and Rfree is the Rfactor for the test set of reflections not used during refinement (5% data set).19.8/24.8R.m.s. bonds (Å)0.007R.m.s. angles (°)1.3a Rsym = Σhkl Σj|Ij(hkl) - 〈Ij(hkl)〉|/Σhkl ΣjI(hkl), where Ij(hkl) and 〈Ij(hkl)〉 are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.b Rfactor = Σ|Fobs - kFcalc|/ΣhklFobs, where k is a scale factor and Rfree is the Rfactor for the test set of reflections not used during refinement (5% data set). Open table in a new tab Structure Determination—We determined the crystal structure of A. aeolicus TadA at a 1.8-Å resolution by the molecular replacement method. The crystal contains four molecules, A, B, C, and D, per asymmetric unit (Fig. 3, A and B). The root mean square deviations of all of the atoms are 1.14, 1.06, and 0.75 Å when comparing A with B, C, and D, respectively. The monomer structure (Fig. 3A) consists of a central β-sheet (β1–β5) with two α-helices (α1, α5) on one side of the sheet and three α-helices (α2–α4) on the other side. Α long loop exists between the β4 and β5 strands and is designated as the β4–β5 loop (residues Lys104-Arg124). Zinc Ion—In the Fo – Fc Fourier map contoured at a 9-σ level, each of the monomers contained one strong spherical density, which was assigned as a zinc ion based on the x-ray absorption fine structure data (Fig. 4). Because no zinc ion was added in the buffer during the protein purification and crystallization, A. aeolicus TadA contains endogenous zinc ions. The zinc ion is tetrahedrally coordinated (Fig. 3, A and C) by His52 Nδ1 (2.1 Å), Cys82 Sγ (2.3 Å), Cys85 Sγ (2.3 Å), and a water molecule, Water O (2.3 Å). Glu54 O∈ interacts with the zinc-bound water (2.5 Å) (Fig. 3, A and C). These residues are conserved among the CDA superfamily members (Fig. 2). The active-site architectures are also similar within the CDA superfamily (8Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar, 9Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar), and the deamination mechanisms may be the same. The zinc ion and Glu54 are proposed to activate the zinc-bound water to form a hydroxide ion. Glu54 may shuttle a proton from the water to the adenosine residue. The zinc-bound hydroxide ion is proposed to attack the C6 atom of the adenosine residue (Fig. 1A) nucleophilically. Dimerization State of TadA—In the crystal, molecules A-B and molecules C-D form two apparent dimers along the noncrystallographic 2-fold axes (Fig. 3B). Each dimer is almost spherical with the exception of the C-terminal protrusions and the zinc-containing cavities (Fig. 3D). The dimerization interface is mainly composed of helices α3 and α4 and the β4–β5 loop. The interface is extensive and buries 1300 Å2 of the total monomer surface area of 8100 Å2. Thirty residues are involved in the dimerization including eight conserved hydrophobic residues (Met55, Ile58, Met84, Ala88, Val112, Phe113, Ile115, and Leu121). In addition, four intersubunit salt bridges are formed between Glu44 O∈ and Lys68 Nζ (2.5 Å) and between Asp48 Oδ and Lys59 Nζ (2.7 Å). When Asp64 in E. coli TadA, which corresponds to Asp48 in A. aeolicus TadA, was replaced by Glu, the mutant enzyme was fully active in vivo but lost its activity in vitro (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar, 25Poulsen L.K. Larsen N.W. Molin S. Andersson P. Mol. Microbiol. 1992; 6: 895-905Crossref PubMed Scopus (27) Google Scholar). This residue may contribute to the structural stabilization of the protein, either by itself or through interaction with other protein(s). The A-B and C-D dimers touch (Fig. 3B) through the A-C, A-D, and B-D contacts of 140, 730, and 150 Å2, respectively, of the 8100-Å2 monomer surface area. Six residues of the C-terminal helix α5 of molecule A interact with nine residues on the wall of the zinc-containing cavity of molecule D and vice versa. An E. coli TadA mutant lacking the C-terminal 17 residues reportedly retained its editing activity (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar, 25Poulsen L.K. Larsen N.W. Molin S. Andersson P. Mol. Microbiol. 1992; 6: 895-905Crossref PubMed Scopus (27) Google Scholar). Therefore, the seven corresponding residues at the C terminus of A. aeolicus TadA may be dispensable for editing (Fig. 2), indicating that the interaction between molecules A and D is not biologically important. All of the interactions between the A-B and C-D dimers may be because of crystal packing. Comparisons of the A. aeolicus TadA Structure to Those of Other CDA Superfamily Members—Sequence alignments (Fig. 2) reveal that the N-terminal halves are conserved among the CDA superfamily members. Residues 1–100 of A. aeolicus TadA share sequence identities of 40% with residues 1–103 of Bacillus subtilis guanine deaminase (GD), 24% with residues 9–111 of yeast CD, 16% with residues 1–106 of B. subtilis CDA, and 15% with residues 9–116 of yeast CDD1. Yeast CDD1 deaminates both mRNA and cytidine (17Dance G.S. Beemiller P. Yang Y. Mater D.V. Mian I.S. Smith H.C. Nucleic Acids Res. 2001; 29: 1772-1780Crossref PubMed Scopus (29) Google Scholar). The C-terminal halves have no sequence conservation. A structural homology search by DALI (26Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (596) Google Scholar) shows that A. aeolicus TadA displays similarities to yeast CD, B. subtilis GD (Protein Data Bank code 1TIY), B. subtilis CDA (Protein Data Bank code 1JTK), and yeast CDD1 (Protein Data Bank code 1R5T) with Z-scores of 20.2, 16.8, 9.7, and 9.6, respectively. Especially, the N-terminal halves, which are shown in pink (Figs. 5 and 6), share high structural similarity. The root mean square deviations of the N-terminal Cα atoms of yeast CD, B. subtilis GD, B. subtilis CDA, and yeast CDD1, superposed on the corresponding residues of A. aeolicus TadA, are 2.55, 1.89, 3.17, and 3.30 Å, respectively. This high structural conservation of the N-terminal motifs would be necessary to coordinate a zinc ion in the active site.Fig. 6Comparison of the monomer structures among the CDA superfamily members. The monomer architectures of A. aeolicus TadA (A), yeast CD (B), B. subtilis GD (C), B. subtilis CDA (D), and yeast CDD1 (E) are shown as ribbon models. The conserved N-terminal motifs are colored pink, and the diverse C-terminal motifs are colored green. As B. subtilis GD forms an intertwined dimer, the C-terminal motif (residues 131–150) of the adjacent subunit and its secondary structure are shown in blue. The zinc ions are represented as magenta spheres.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next describe the comparisons of the oligomerization states and of the C-terminal halves of architectures. A. aeolicus TadA (Fig. 5A) and yeast CD (Fig. 5B) form similar dimeric structures. Their dimer interfaces comprise helices α3 and α4 and the regions between β4 and β5 (the loop in A. aeolicus TadA and the helix α5 in yeast CD). In addition, the C-terminal motifs of the monomers have similar structures (Fig. 6). First, the β4 and β5 strands run parallel to each other. Second, the N and C termini are located on the same side of the α/β/α three-layered fold. Third, both of the C-terminal helices (α5 in A. aeolicus TadA and α6 in yeast CD) contact the α1 N-terminal helices. B. subtilis GD forms an intertwined dimer through C-terminal domain swapping (Fig. 5C). The dimer interface is composed of the helices α3 and α4 and the C-terminal domain. Helices α5 and α6, which are located between the β4 and β5 strands, make the domain swapping possible and have important roles in guanine recognition (11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). As a consequence of the swap, the C-terminal motif of one subunit interacts with the other subunit in a manner similar to that of A. aeolicus TadA and yeast CD (Fig. 6C). Yeast CDD1 and B. subtilis CDA form tetrameric structures. One subunit of yeast CDD1 (Fig. 5D) interacts with the other three subunits via the α2–α6 helices. B. subtilis CDA also has a tetrameric structure similar to that of yeast CDD1. The tetramers are not formed by the dimerization of two TadA-like dimers. Therefore, the quaternary structure of yeast CDD1 (Fig. 5D) completely differs from that of A. aeolicus TadA (Fig. 5A). Also, the C-terminal motifs of B. subtilis CDA (Fig. 6D) and yeast CDD1 (Fig. 6E) share no structural similarities with that of A. aeolicus TadA (Fig. 6A). First, the β4 and β5 strands run antiparallel to each other and only short turns exist between the β4 and β5 strands. Second, the N and C termini are located on the opposite sides of the α/β/α three-layered fold. The oligomerization state does not determine whether nucleic acids are accommodated. Based on the structure of yeast CDD1, a model of human activation-induced cytidine deaminase has been made (12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar) on the assumption that its quaternary structure is similar to a tetramer. On the other hand, an analysis of the human activation-induced cytidine deaminase sequence with the 3DJury method suggested that the dimeric yeast CD was the best template (27Zaim J. Kierzek A.M. Nat. Immunol. 2003; 4: 1153-1154Crossref PubMed Scopus (10) Google Scholar). One problem with using yeast CD as a template was that the intramolecular active site was too small to accommodate large nucleic acid molecules (12Xie K. Sowden M.P. Dance G.S. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar). However, the structure of A. aeolicus TadA indicates that activation-induced cytidine deaminase might form a TadA-like dimer for nucleic acid binding. The RNA-binding Site of A. aeolicus TadA—We next examined the substrate-binding site differences between A. aeolicus TadA and yeast CD to determine which motifs contribute to the specific binding of their substrates, tRNA and cytosine, respectively. The active-site cavity of A. aeolicus TadA (Fig. 5A) is composed of the α2 and α5 helices, and the zinc ion from one subunit, the β4–β5 loops from both subunits, and loop 1 (between α2 and β3) and loop 2 (between α3 and β4) from the other subunit. Both of the β4–β5 loops of the A. aeolicus TadA dimer are extended and cooperate with each other to form the putative RNA-binding site (Fig. 5A). On the other hand, the corresponding regions (the α5 helices) in the yeast CD dimer are involved in the intersubunit interaction but are far from the cytosine-binding site (Fig. 5B). The C-terminal α5 helix of A. aeolicus TadA protrudes outward, whereas the corresponding C-terminal α7 helix of yeast CD bends inward, which makes the cavity narrower and suitable for a small cytosine base (10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In summary, the diversified regions between the β4 and β5 strands and the C-terminal helices have important roles in the specific recognition of the substrates by A. aeolicus TadA and yeast CD. The Recognition of tRNAArg-2—The anticodon stem loop structure (Fig. 1B) is reportedly sufficient for E. coli TadA to deaminate the adenosine residue at the wobble position (position 34) (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar). A mutational study showed that the bases 33–36 (UACG) are recognized base-specifically and that the stem is recognized base-nonspecifically (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar). Fig. 7A shows the side chains of the conserved residues in the active-site cavity of A. aeolicus TadA, which should hold the anticodon loop of tRNA so that the adenosine residue lies just above the zinc-bound hydroxide ion. The asparagine residue at the C-terminal end of the β2 strand is widely conserved within the CDA superfamily (Fig. 2) and is known to interact with cytidine, cytosine, and guanine (9Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 10Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The corresponding residue, Asn41, in A. aeolicus TadA is located at the bottom of the active site and may contribute to the recognition of the adenosine residue at position 34. The adenosine residue normally base-stacks with nucleotide residues 35–38 but should be unstacked for the deamination. The conserved hydrophobic residues (Val23, Val25, Val77, Leu79, and Leu139) colored yellow in Fig. 7A cluster on the wall of the cavity and may accommodate the unstacked anticodon. The stem moiety may be recognized by the helix α5, which includes some conserved positively charged amino acid residues (Fig. 2). Lys144 and Arg147 in A. aeolicus TadA lie on the same side of the helix (Fig. 7A). We thought that these positively charged side chains may interact with the negatively charged tRNA phosphate backbone. The seven C-terminal residues including Arg147 in A. aeolicus TadA are reportedly not essential for editing (7Wolf J. Gerber A.P. Keller W. EMBO J. 2002; 21: 3841-3851Crossref PubMed Scopus (172) Google Scholar, 25Poulsen L.K. Larsen N.W. Molin S. Andersson P. Mol. Microbiol. 1992; 6: 895-905Crossref PubMed Scopus (27) Google Scholar). Thus, as long as TadA has Lys144, it may be able to bind with tRNA. Fig. 7, B and C, show a preliminary docking model of TadA and the anticodon stem loop moiety (bases 26–43) of yeast tRNAPhe (Protein Data Bank code 1EHZ). We docked them with no conformational change. The anticodon stem loop is well embedded in the putative RNA binding cavity (Fig. 7B). The surface of A. aeolicus TadA is color-coded according to its electrostatic potential (red, –10kT/e; blue, +10kT/e) (Fig. 7C). The positive charges at the C-terminal helix are located near the tRNA phosphate backbones. Several conserved residues exist at the entrance of the cavity (Fig. 7A). Lys105, Phe142, and Phe143 of one subunit and Lys68, Tyr69, Arg93, His123, and Asn124 from the other subunit may also interact with the tRNA. Lys68 and Tyr69 are conserved as a combination of a positively charged residue and an aromatic residue (Fig. 2). The side chains of these two residues are fixed by a cation-π interaction. The conserved Arg93 residue is also located adjacent to Tyr69 in A. aeolicus TadA. These residues may contribute to specific interactions with the anticodon bases. In the loop region, Asn122 and His123 are not very close to the putative catalytic site but the loop might change its conformation and bring them closer upon tRNA binding. In summary, the large cavity formed around the catalytic site with the zinc ion seems to be important for accommodating the anticodon loop stem of tRNAArg-2. This structural feature clearly distinguishes TadA from yeast cytosine deaminase, which has a smaller substrate. We thank Dr. M. Yamamoto for technical assistance at the beamline BL26B1 of SPring-8 and Drs. T. Yanagisawa and R. Ishitani for technical advice regarding the protein purification. We thank T. Kobayashi for critically reading the manuscript and T. Nakayama and K. Yajima for help in preparing the manuscript." @default.
• W2074731176 created "2016-06-24" @default.
• W2074731176 creator A5012045946 @default.
• W2074731176 creator A5017102088 @default.
• W2074731176 creator A5027781657 @default.
• W2074731176 creator A5034100486 @default.
• W2074731176 creator A5047650631 @default.
• W2074731176 creator A5055954909 @default.
• W2074731176 creator A5058452895 @default.
• W2074731176 creator A5073442482 @default.
• W2074731176 date "2005-04-01" @default.
• W2074731176 modified "2023-10-04" @default.
• W2074731176 title "Crystal Structure of tRNA Adenosine Deaminase (TadA) from Aquifex aeolicus" @default.
• W2074731176 cites W1539796472 @default.
• W2074731176 cites W1590413499 @default.
• W2074731176 cites W1639542273 @default.
• W2074731176 cites W1966501160 @default.
• W2074731176 cites W1970257155 @default.
• W2074731176 cites W1973732415 @default.
• W2074731176 cites W1985629904 @default.
• W2074731176 cites W1986191025 @default.
• W2074731176 cites W1986851354 @default.
• W2074731176 cites W2001641653 @default.
• W2074731176 cites W2011374678 @default.
• W2074731176 cites W2013083986 @default.
• W2074731176 cites W2021484484 @default.
• W2074731176 cites W2038989483 @default.
• W2074731176 cites W2045537814 @default.
• W2074731176 cites W2047173579 @default.
• W2074731176 cites W2063337242 @default.
• W2074731176 cites W2068694787 @default.
• W2074731176 cites W2073655863 @default.
• W2074731176 cites W2083691392 @default.
• W2074731176 cites W2094617711 @default.
• W2074731176 cites W2097382368 @default.
• W2074731176 cites W2097493124 @default.
• W2074731176 cites W2119314909 @default.
• W2074731176 cites W2129334819 @default.
• W2074731176 cites W2131423362 @default.
• W2074731176 cites W2139387072 @default.
• W2074731176 cites W2155479906 @default.
• W2074731176 cites W4234887284 @default.
• W2074731176 cites W2034937673 @default.
• W2074731176 doi "https://doi.org/10.1074/jbc.m414541200" @default.
• W2074731176 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15677468" @default.
• W2074731176 hasPublicationYear "2005" @default.
• W2074731176 type Work @default.
• W2074731176 sameAs 2074731176 @default.
• W2074731176 citedByCount "48" @default.
• W2074731176 countsByYear W20747311762012 @default.
• W2074731176 countsByYear W20747311762013 @default.
• W2074731176 countsByYear W20747311762014 @default.
• W2074731176 countsByYear W20747311762015 @default.
• W2074731176 countsByYear W20747311762016 @default.
• W2074731176 countsByYear W20747311762017 @default.
• W2074731176 countsByYear W20747311762018 @default.
• W2074731176 countsByYear W20747311762019 @default.
• W2074731176 countsByYear W20747311762020 @default.
• W2074731176 countsByYear W20747311762021 @default.
• W2074731176 countsByYear W20747311762022 @default.
• W2074731176 crossrefType "journal-article" @default.
• W2074731176 hasAuthorship W2074731176A5012045946 @default.
• W2074731176 hasAuthorship W2074731176A5017102088 @default.
• W2074731176 hasAuthorship W2074731176A5027781657 @default.
• W2074731176 hasAuthorship W2074731176A5034100486 @default.
• W2074731176 hasAuthorship W2074731176A5047650631 @default.
• W2074731176 hasAuthorship W2074731176A5055954909 @default.
• W2074731176 hasAuthorship W2074731176A5058452895 @default.
• W2074731176 hasAuthorship W2074731176A5073442482 @default.
• W2074731176 hasBestOaLocation W20747311761 @default.
• W2074731176 hasConcept C104317684 @default.
• W2074731176 hasConcept C153957851 @default.
• W2074731176 hasConcept C185592680 @default.
• W2074731176 hasConcept C2776991684 @default.
• W2074731176 hasConcept C2777400929 @default.
• W2074731176 hasConcept C34628245 @default.
• W2074731176 hasConcept C547475151 @default.
• W2074731176 hasConcept C55493867 @default.
• W2074731176 hasConcept C67705224 @default.
• W2074731176 hasConceptScore W2074731176C104317684 @default.
• W2074731176 hasConceptScore W2074731176C153957851 @default.
• W2074731176 hasConceptScore W2074731176C185592680 @default.
• W2074731176 hasConceptScore W2074731176C2776991684 @default.
• W2074731176 hasConceptScore W2074731176C2777400929 @default.
• W2074731176 hasConceptScore W2074731176C34628245 @default.
• W2074731176 hasConceptScore W2074731176C547475151 @default.
• W2074731176 hasConceptScore W2074731176C55493867 @default.
• W2074731176 hasConceptScore W2074731176C67705224 @default.
• W2074731176 hasIssue "16" @default.
• W2074731176 hasLocation W20747311761 @default.
• W2074731176 hasOpenAccess W2074731176 @default.
• W2074731176 hasPrimaryLocation W20747311761 @default.
• W2074731176 hasRelatedWork W1498415432 @default.
• W2074731176 hasRelatedWork W1504841080 @default.
• W2074731176 hasRelatedWork W1971449617 @default.
• W2074731176 hasRelatedWork W2016119250 @default.
• W2074731176 hasRelatedWork W2016866166 @default.
• W2074731176 hasRelatedWork W2035955540 @default.

• next