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• W2034304005 abstract "ADP-ribose pyrophosphatase (ADPRase) catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose to ribose 5′-phosphate and AMP. This enzyme plays a key role in regulating the intracellular ADP-ribose levels, and prevents nonenzymatic ADP-ribosylation. To elucidate the pyrophosphatase hydrolysis mechanism employed by this enzyme, structural changes occurring on binding of substrate, metal and product were investigated using crystal structures of ADPRase from an extreme thermophile, Thermus thermophilus HB8. Seven structures were determined, including that of the free enzyme, the Zn2+-bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADP-ribose and Zn2+ or Gd3+, and the product complexes with AMP and Mg2+ or with ribose 5′-phosphate and Zn2+. The structural and functional studies suggested that the ADP-ribose hydrolysis pathway consists of four reaction states: bound with metal (I), metal and substrate (II), metal and substrate in the transition state (III), and products (IV). In reaction state II, Glu-82 and Glu-70 abstract a proton from a water molecule. This water molecule is situated at an ideal position to carry out nucleophilic attack on the adenosyl phosphate, as it is 3.6 Å away from the target phosphorus and almost in line with the scissile bond. ADP-ribose pyrophosphatase (ADPRase) catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose to ribose 5′-phosphate and AMP. This enzyme plays a key role in regulating the intracellular ADP-ribose levels, and prevents nonenzymatic ADP-ribosylation. To elucidate the pyrophosphatase hydrolysis mechanism employed by this enzyme, structural changes occurring on binding of substrate, metal and product were investigated using crystal structures of ADPRase from an extreme thermophile, Thermus thermophilus HB8. Seven structures were determined, including that of the free enzyme, the Zn2+-bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADP-ribose and Zn2+ or Gd3+, and the product complexes with AMP and Mg2+ or with ribose 5′-phosphate and Zn2+. The structural and functional studies suggested that the ADP-ribose hydrolysis pathway consists of four reaction states: bound with metal (I), metal and substrate (II), metal and substrate in the transition state (III), and products (IV). In reaction state II, Glu-82 and Glu-70 abstract a proton from a water molecule. This water molecule is situated at an ideal position to carry out nucleophilic attack on the adenosyl phosphate, as it is 3.6 Å away from the target phosphorus and almost in line with the scissile bond. Nudix pyrophosphatases are widely distributed in nature and share a highly conserved amino acid sequence motif called the “Nudix motif” (GX5EX7REUXEEXGU, where U is one of the bulky hydrophobic amino acids I, L, or V), which adopts a unique loop-helix-loop structure (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Enzymes in this family catalyze the hydrolysis of nucleoside diphosphates, linked to another moiety x. Their postulated role is to control the cellular concentration of toxic nucleoside diphosphate derivatives or physiological metabolites, accumulation of which could be harmful (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). ADP-ribose (ADPR) 1The abbreviations used are: ADPR, ADP-ribose; ADPRase, ADP-ribose pyrophosphatase; EcADPRase, E. coli ADP-ribose pyrophosphatase; MAD, multiwavelength anomalous diffraction; MtADPRase, M. tuberculosis ADP-ribose pyrophosphatase; OAADPR, O-acetyl-ADP-ribose; Se-Met, selenomethionyl; TtADPRase, T. thermophilus ADP-ribose pyrophosphatase.1The abbreviations used are: ADPR, ADP-ribose; ADPRase, ADP-ribose pyrophosphatase; EcADPRase, E. coli ADP-ribose pyrophosphatase; MAD, multiwavelength anomalous diffraction; MtADPRase, M. tuberculosis ADP-ribose pyrophosphatase; OAADPR, O-acetyl-ADP-ribose; Se-Met, selenomethionyl; TtADPRase, T. thermophilus ADP-ribose pyrophosphatase. is one such diphosphate derivative, which is produced enzymatically as part of the turnover of NAD+, cyclic ADPR, ADP-ribosylated proteins, and poly-ADP-ribosylated proteins. Although certain proteins are posttranslationally modified by ADPR, high intracellular levels of ADPR could result in nonenzymatic ADP-ribosylation. This is a deleterious process that inactivates enzymes and could interfere with the recognition of enzymatic ADP-ribosylation (2Just I. Wollenberg P. Moss J. Aktories K. Eur. J. Biochem. 1994; 221: 1047-1054Crossref PubMed Scopus (35) Google Scholar). ADPR pyrophosphatases (ADPRases) catalyze the hydrolysis of ADPR to AMP and ribose 5′-phosphate to prevent ADPR accumulation. ADPRase activity has been detected in all three kingdoms (3Dunn C.A. O'Handley S.F. Frick D.N. Bessman M.J. J. Biol. Chem. 1999; 274: 32318-32324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 4Gasmi L. Cartwright J.L. McLennan A.G. Biochem. J. 1999; 344: 331-335Crossref PubMed Google Scholar, 5O'Handley S.F. Frick D.N. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 3192-3197Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 6Sheikh S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 7Ribeiro J.M. Costas M.J. Cameselle J.C. J. Biochem. Mol. Toxicol. 1999; 13: 171-177Crossref PubMed Google Scholar), but the specificity for ADPR over other substrates and the selectivity of metal ions required for activity vary between species. The mechanism underlying the different substrate specificity and the metal dependence is unknown at both the structural and functional levels. Elucidation of these properties requires the study of ADPRases from numerous sources. In this article, we investigated the catalytic mechanism of ADPRase from an extreme thermophile, Thermus thermophilus HB8 (TtADPRase). In general, proteins isolated from T. thermophilus are heat-stable and suitable for physicochemical studies, including x-ray crystallography (8Yokoyama S. Hirota H. Kigawa T. Yabuki T. Shirouzu M. Terada T. Ito Y. Matsuo Y. Kuroda Y. Nishimura Y. Kyogoku Y. Miki K. Masui R. Kuramitsu S. Nat. Struct. Biol. 2000; 7: 943-945Crossref PubMed Scopus (325) Google Scholar, 9Yokoyama S. Matsuo Y. Hirota H. Kigawa T. Shirouzu M. Kuroda Y. Kurumizaka H. Kawaguchi S. Ito Y. Shibata T. Kainosho M. Nishimura Y. Inoue Y. Kuramitsu S. Prog. Biophys. Mol. Biol. 2000; 73: 363-376Crossref PubMed Scopus (47) Google Scholar). TtADPRase catalyzes the hydrolysis of ADPR to AMP and ribose 5′-phosphate in the presence of Mg2+ and Zn2+ ions. The enzyme is also heat-stable and retains ADPRase activity at 75 °C. The structures of the free enzyme, the Zn2+-bound enzyme, the binary complex with ADPR, the ternary complexes with ADPR and metal, and the product complexes containing AMP or ribose 5′-phosphate were determined. Based on structural and functional analyses, we propose a novel reaction mechanism for ADPR pyrophosphate hydrolysis that is different from those proposed based on the ternary complex structures of ADPRases from Escherichia coli (EcADPRase) and from Mycobacterium tuberculosis (MtADPRase) (10Gabelli S.B. Bianchet M.A. Ohnishi Y. Ichikawa Y. Bessman M.J. Amezel L.M. Biochemistry. 2002; 41: 9279-9285Crossref PubMed Scopus (62) Google Scholar, 11Kang L.-W. Gabelli S.B. Cunningham J.E. O'Handley S.F. Amzel L.M. Structure. 2003; 11: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Preparation of Mutant TtADPRases—pT7Blue-ndx4, which contains the ndx4 gene, was used as a template of mutagenesis (12Yoshiba S. Nakagawa N. Masui R. Shibata T. Inoue Y. Yokoyama S. Kuramitsu S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1840-1842Crossref PubMed Scopus (7) Google Scholar). pT7Bluendx4 was digested with BamHI and HindIII, and the fragment was inserted into pKF19K (Takara Shuzo Co., Kyoto, Japan) digested with the same enzymes. Site-directed mutagenesis of E82Q, E86Q, and E129Q was performed according to the Mutan-Super Express Km kit instruction manual (Takara Shuzo). Site-directed mutagenesis of D126N, E127Q, and D128N was performed by replacement of the wild-type sequence in pT7Blue-ndx4 plasmid with each mutated fragment. E82Q mutant fragment was inserted into pET-3a, whereas the other mutant fragments were inserted into pET-11a. Overproduction and purification of mutant proteins were carried out in a similar manner to that for wild-type enzyme (12Yoshiba S. Nakagawa N. Masui R. Shibata T. Inoue Y. Yokoyama S. Kuramitsu S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1840-1842Crossref PubMed Scopus (7) Google Scholar). Enzyme Assays—TtADPRase activities of wild-type, E82Q mutant, and E86Q mutant were analyzed by reversed-phase high performance liquid chromatography. The reaction solution contained 50 mm Tris-HCl, pH 7.5, 5 mm MgCl2 or 250 μm ZnSO4, 0.1 m KCl, 50 nm wild-type enzyme or 5.5 μm for E82Q and E86Q mutants, and various concentrations of ADPR. The 100-μl reaction mixture was incubated at 25 °C for 3 min for the wild-type or for 11 h for the E82Q and E86Q mutants. Reactions were stopped by the addition of an equivalent volume of 100 mm EDTA. The mixture was applied to a C18 column (CAPCELL PAK C18 MG column, Shiseido, Tokyo, Japan) equilibrated with 20 mm sodium phosphate, pH 7.0, 5 mm tetrabutylammonium phosphate and 5% (v/v) methanol with AKTA explorer (Amersham Biosciences). The elution was performed at 1 ml min-1 with a 5-50% gradient of methanol in the equilibration buffer, and an absorbance at 260 nm was monitored. AMP and ADPR were eluted at elution volumes of 5 and 10 ml, respectively. The amounts of compounds were assessed from their peak areas. TtADPRase activities of wild-type, D126N, E127Q, D128N, and E129Q mutants were analyzed by measuring inorganic orthophosphate production using the colorimetric procedure of Ames and Dubin (13Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar). The 100-μl reaction solution contained 50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 0.1 m KCl, 50 nmTtADPRase, 2 units of calf intestine alkaline phosphatase, and various concentrations of ADPR. After incubation at 25 °C for 3 min, 75 μl of reaction mixture was mixed with 175 μl of the ascorbic-molybdate mixture and incubated at 45 °C for 20 min. Then, the quantity of phosphoric acid was measured at an absorbance of 820 nm. The kinetic parameters were obtained by fitting the initial rate of product formation to the Michaelis-Menten equation. Crystallization—The method for crystallization and data collection of the free enzyme and the binary complex has been described previously (12Yoshiba S. Nakagawa N. Masui R. Shibata T. Inoue Y. Yokoyama S. Kuramitsu S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1840-1842Crossref PubMed Scopus (7) Google Scholar). Overproduction of selenomethionyl (Se-Met) TtADPRase was performed as follows. E. coli B834(DE3) cells carrying the pET-11a-ndx4 plasmid were cultured in LB medium at 37 °C for a few hours. The preculture was incubated at a dilution ratio of 100:1 (v/v) into LeMaster medium containing Se-Met with lactose as a carbon source and cultured at 37 °C for 24 h. The Se-Met TtADPRase was purified in a manner similar to that for native protein (12Yoshiba S. Nakagawa N. Masui R. Shibata T. Inoue Y. Yokoyama S. Kuramitsu S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1840-1842Crossref PubMed Scopus (7) Google Scholar). All crystals, except the binary complex form, were obtained under the same condition as that of the free enzyme, with slight adjustments in pH or the concentration of ammonium sulfate, and the addition of metal, substrate, or product. Crystals were flash-cooled in a nitrogen stream, and data were collected under cryo conditions. Multiwavelength Anomalous Diffraction (MAD) Phasing, Model Building, and Refinement of the Free Enzyme—MAD data were collected at four wavelengths near the selenium absorption edge at the beamline BL44B2 at SPring-8 (14Adachi S. Oguchi T. Tanida H. Park S.-Y. Shimizu H. Miyatake H. Kamiya N. Shiro Y. Inoue Y. Ueki T. Iizuka T. Nucl. Instrum. Methods Phys. Res. 2001; A467/A468: 711-714Crossref Scopus (59) Google Scholar). Diffraction images were processed using the HKL2000 program (15Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The structure was determined by the MAD method using programs from the CCP4 suite (16Collaborative Computational Project No 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) and data collected at four wavelengths (edge, high remote, low remote, and peak). The initial model was built with the aid of the amino acid sequence using the program O (17Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar), and refined from 15- to 2.5-Å resolution using the phase information from the diffraction data with the CNS program (18Brünger A.T. Adams P.D. Clore G.M. DeLano W.N. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). The three selenium atom sites deduced from the difference Fourier map coincided with the three sulfur sites of the methionine residues in the model. The N-terminal region was disordered, and the first Met was not detected. Progress in the structural refinement was evaluated at each stage by the free R factor and by inspection of stereochemical parameters calculated by the PROCHECK program (19Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Final phasing and refinement statistics are shown in Table I.Table IStatistics for data and refinement of the Se-Met derivative and free enzymeData collectionData set nameSe-MetFree enzymePeakEdgeHigh remoteLow remoteSpace groupP3221P3221Cell constant (Å)a = b = 49.8, c = 118.5a = b = 49.6, c = 117.9Wavelength (Å)0.98040.98080.97630.98221.0Resolution (Å)aValues in parentheses refer to the highest resolution shell.50−1.70 (1.76−1.70)50−1.70 (1.76−1.70)50−1.70 (1.76−1.70)50−1.70 (1.76−1.70)50−1.50 (1.55−1.50)No. of observations195,717195,671195,663195,38340,836Unique reflections19,43619,43019,43119,45726,995Data completeness (%)99.8 (100)99.7 (100)99.8 (100)99.8 (100)97.2 (93.5)Mean I/σI38.3 (6.1)38.6 (6.1)38.3 (6.2)38.8 (6.3)34.3 (3.6)Rsym (%)bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.5.4 (21.0)4.4 (20.2)4.7 (19.9)3.6 (19.7)3.3 (25.0)Phasing power acentriccPhasing power acentric = mean FH/lack of closure.3.56Cullis R acentricdCullis R acentric = lack of closure/isomorphous difference.0.58Model refinement statistics (resolution for refinement)(50−1.76)RcrysteRcryst=∑||Fobs|−|Fcal||/∑|Fobs|./RfreefRfree is monitored with 10% of the reflection data excluded from refinement.20.9/23.1Average B factor17.23Deviations from idealityBond lengths (Å)0.005Bond angles (°)1.25Ramachandran plot Favored88.9Additional allowed11.1a Values in parentheses refer to the highest resolution shell.b Rsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.c Phasing power acentric = mean FH/lack of closure.d Cullis R acentric = lack of closure/isomorphous difference.e Rcryst=∑||Fobs|−|Fcal||/∑|Fobs|.f Rfree is monitored with 10% of the reflection data excluded from refinement. Open table in a new tab Data Collection and Refinement of the Wild-type Complexes—The crystals of the Zn2+-bound enzyme were obtained by mixing 1:1 the reservoir solution and the solution containing 2 mm protein and 2 mm ZnCl2. Data were collected at the wavelengths of peak (1.278 Å) and remote (1.292 Å) at the beamline BL44B2 at SPring-8. Diffraction images were processed using the HKL2000 program. Data collection and refinement statistics are shown in Table II.Table IIStatistics for data and refinement of wild-type complexesData set nameData collectionZn2+-bound enzyme (ligand Zn2+)Binary complex (ligand ADPR)Zn2+ ternary complex (ligands Zn2+ and ADPR)PeakLow remotePeakLow remoteSpace groupP3221P3221P3221Cell constant (Å)a = b = 49.9, c = 118.31a = b = 49.8, c = 119.3a = b = 49.7, c = 118.9Wavelength (Å)1.2781.2921.541.2821.287Resolution (Å)aValues in parentheses refer to the highest resolution shell.50−1.60 (1.66−1.60)50−1.6019−2.01 (2.12−2.01)20−1.8 (1.86−1.80)20−1.8No. of observations333,276323,773164,512192,144190,720Unique reflections23,02823,01211,98630,06230,068Data completeness (%)99.0 (92.4)98.7 (91.5)99.9 (99.9)99.2 (99.2)99.1 (98.1)Mean I/σI57.2 (3.9)60.7 (4.6)11.2 (10.3)35.26 (12.8)35.36 (12.5)Rsym (%)bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.2.7 (22.4)2.4 (20.0)4.0 (5.7)4.9 (8.9)4.8 (8.6)Model refinement statisticsResolution for refinement43−1.7419−2.020−1.8RcrystcRcryst=∑||Fobs|−|Fcal||/∑|Fobs|. /RfreedRfree is monitored with 10% of the reflection data excluded from refinement.22.1/25.621.3/23.420.6/23.5Average B factor22.5817.5921.45Deviations from idealityBond lengths (Å)0.0040.0130.004Bond angles (°)1.282.021.28Ramachandran plotFavored91.391.091.5Additional allowed8.79.08.5Reaction state in Fig. 8IIIIIData set nameData collectionGd3+ ternary complex (ligands Gd3+ and ADPR)PeakLow remoteHigh remoteSpace groupP3221P3221Cell constant (Å)a = b = 49.6, c = 119.8a = b = 49.8, c = 119.3Wavelength (Å)1.7111.7161.00Resolution (Å)50−2.3 (2.38−2.30)50−2.350−1.8 (1.86−1.80)No. of observations104,941105,462234,961Unique reflections8,0548,06316,475Data completeness (%)99.5 (98.1)99.5 (98.1)99.8 (100)Mean I/σI23.7 (18.0)40.6 (11.4)24.6 (3.8)Rsym (%)bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.9.7 (19.2)5.0 (13.7)6.2 (28.3)Model refinement statisticsResolution for refinement50−1.8RcrystcRcryst=∑||Fobs|−|Fcal||/∑|Fobs|. /RfreedRfree is monitored with 10% of the reflection data excluded from refinement.22.3/24.6Average B factor22.70Deviations from idealityBond lengths (Å)0.005Bond angles (°)1.29Ramachandran plotFavored90.4Additional allowed9.6Reaction state in Fig. 8IIData collectionData set nameProduct complex I (ligands AMP and Mg2+)Product complex II (ligands ribose 5′-phosphate and Zn2+)Space groupP3221P3221Cell constant (Å)a = b = 49.7, c = 118.1a = b = 49.7, c = 118.0Wavelength (Å)1.541.54Resolution (Å)39.37−2.2 (2.28−2.20)40−2.6 (2.69−2.60)No. of observations36,24141,850Unique reflections8,3025,224Data completeness (%)91.1 (85.4)93.4 (85.6)Mean I/σI16.9 (10.5)25.3 (17.3)Rsym (%)bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.2.9 (6.8)4.0 (5.7)Model refinement statisticsResolution for refinement39.37−2.2040−2.6RcrystcRcryst=∑||Fobs|−|Fcal||/∑|Fobs|. /RfreedRfree is monitored with 10% of the reflection data excluded from refinement.19.9/24.822.0/24.5Average B factor19.7421.37Deviations from idealityBond lengths (Å)0.0050.007Bond angles (°)1.321.40Ramachandran plotFavored91.489.8Additional allowed8.610.2Reaction state in Fig. 8IVIVa Values in parentheses refer to the highest resolution shell.b Rsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.c Rcryst=∑||Fobs|−|Fcal||/∑|Fobs|.d Rfree is monitored with 10% of the reflection data excluded from refinement. Open table in a new tab The crystals of the ternary complex were obtained by mixing 1:1 the reservoir solution and the solution containing 2 mm protein, 2 mm ADPR, and 2 mm ZnCl2 or GdCl3. X-ray absorption fine structure measurement determined the Zn2+ absorption peak and low remote to be 1.282 and 1.287 Å, respectively, and the Gd3+ absorption peak and low remote to be 1.711 and 1.716 Å, respectively, at the beamline BL44B2 at SPring-8. Then, diffraction data of the ternary complex were collected at these wavelengths. Additionally, data of the Gd3+ ternary complex were also collected at the wavelength of 1.0 Å. The crystals that contained AMP were obtained by mixing 1:1 the reservoir solution and the solution containing 2 mm protein, 10 mm AMP, 2 mm ribose 5′-phosphate, and 2 mm MgCl2. The crystals that contained ribose 5′-phosphate were obtained by mixing 1:1 the reservoir solution and the solution containing 2 mm protein, 2 mm AMP, 2 mm ribose 5′-phosphate, and 2 mm ZnCl2. X-ray diffraction experiments were carried out in the laboratory using Cu Kα radiation. Data were collected with R-Axis VII (Rigaku) and were processed with Crystal-Clear 1.3.5 (Molecular Structure Corp., dxTREK version 8.OSSI). The structures were determined by molecular replacement with the AMoRe program (20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar) using the free enzyme as starting model and refined by CNS, and the metal sites were located in anomalous difference Fourier maps. Data Collection and Refinement of Mutants E82Q and E86Q—Crystals of two mutant ternary complexes were produced by mixing 1:1 the reservoir solution and the solution containing 2 mm mutant protein, 2 mm ADPR, and 2 mm MgCl2 or ZnCl2. The x-ray diffraction experiments and data collections of the Mg2+ complexes were carried out in the laboratory in a manner similar to that for the product complex. To identify the Zn2+ positions, data for the E82Q ternary complex with Zn2+ were collected at the wavelengths corresponding to peak (1.282 Å) and remote (1.290 Å) at the beamline BL26B2 at SPring-8 (21Yamamoto M. Goto S. Takeshita K. Ishikawa T. SPring-8 Inf. 2001; 6: 202-206Google Scholar) and data for the E86Q ternary complex with Zn2+ were collected at the wavelengths corresponding to peak (1.278 Å) and remote (1.292 Å) at the beamline BL44B2 at SPring-8. The mutant structures were determined in a manner similar to that for the wild-type ternary complex. The same refinement procedures were applied, and statistics are shown in Table III.Table IIIStatistics for data and refinement of mutantsData collectionData set nameE82Q (ligands Mg2+ and SO2−4)E86Q (ligands Mg2+ and ADPR)Space groupP3221P3221Cell constant (Å)a = b = 49.9, c = 117.9a = b = 49.9, c = 118.1Wavelength (Å)1.541.54Resolution (Å)40.55−1.90 (1.97−1.90)40.55−1.97 (2.04−1.97)No. of observations71,87667,237Unique reflections13,32212,121Data completeness (%)95.1 (89.3)96.1 (91.1)Mean I/σI38.7 (24.2)38.8 (26.3)Rsym (%)aValues in parentheses refer to the highest resolution shell., bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.2.4 (3.4)2.3 (3.8)Model refinement statisticsRcrystcRcryst=∑||Fobs|−|Fcal||/∑|Fobs| /RfreedRfree is monitored with 10% of the reflection data excluded from refinement.20.3/22.121.2/22.7Average B factor19.7121.7Deviations from idealityBond lengths (Å)0.0050.005Bond angles (°)1.361.28Ramachandran plotFavored93.193.8Additional allowed6.96.2Data set nameData collectionE82Q (ligands Zn2+ and SO2−4)E86Q (ligands Zn2+ and ADPR)PeakLow remotePeakLow remoteSpace groupP3221P3221Cell constant (Å)a = b = 49.9, c = 118.6a = b = 49.7, c = 118.5Wavelength (Å)1.2821.2901.2781.292Resolution (Å)50−1.66 (1.72−1.66)50−1.65 (1.71−1.65)No. of observations475,174479,474318,311301,499Unique reflections22,44622,28421,10321,143Data completeness (%)96.2 (95.8)95.7 (94.7)99.8 (99.7)99.8 (99.0)Mean I/σI66.97 (22.24)67.37 (23.36)54.4 (5.0)54.8 (4.8)Rsym (%)bRsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.4.0 (14.5)3.7 (15.1)3.2 (24.8)3.5 (24.3)Model refinement statisticsRcrystcRcryst=∑||Fobs|−|Fcal||/∑|Fobs| /RfreedRfree is monitored with 10% of the reflection data excluded from refinement.22.1/22.721.6/22.6Resolution for refinement(50−1.66)(43−1.65)Average B factor22.1321.50Deviations from idealityBond lengths (Å)0.0040.004Bond angles (°)1.281.30Ramachandran plotFavored90.091.3Additional allowed10.08.7a Values in parentheses refer to the highest resolution shell.b Rsym=∑hkl∑i|Ihkl,i−〈Ihkl〉|/∑hkl∑iIhkl,i, where 〈Ihkl〉 is the mean intensity of multiple Ihkl,i observations for symmetry-related reflections.c Rcryst=∑||Fobs|−|Fcal||/∑|Fobs|d Rfree is monitored with 10% of the reflection data excluded from refinement. Open table in a new tab Structure diagrams were drawn using the MOLSCRIPT (22Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOB-SCRIPT (23Esnouf R.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar), and Raster3D programs (24Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). In figures, atoms of α- and β-phosphate of ADPR are represented by letters A and B, not by α and β. The dimer structure of the Gd3+ ternary complex of TtADPRase is shown in Fig. 1A. The two identical monomers (one subunit is colored red and blue, and another subunit is colored pink and light blue) were related by a crystallographic 2-fold axis, which was consistent with the result of gel filtration chromatography (12Yoshiba S. Nakagawa N. Masui R. Shibata T. Inoue Y. Yokoyama S. Kuramitsu S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1840-1842Crossref PubMed Scopus (7) Google Scholar). The structure of TtADPRase did not show large conformational change, regardless of whether it exists as the free enzyme, Zn2+-bound enzyme, binary complex with ADPR, ternary complexes with ADPR and Zn2+ or Gd3+, or product complexes with AMP or ribose 5′-phosphate. When the Cα atoms of these six structures were superimposed on the Gd3+ ternary complex structure, the average root mean square deviation values between the Gd3+ ternary complex structure and the other structures ranged from 0.20 to 0.26 Å. The subunit was divided into two distinctive structural domains (Fig. 1, A and B): an N-terminal domain (residues 1-34, colored red) and a C-terminal domain, which is also referred to as the Nudix domain (residues 35-170, colored blue). The N-terminal domain comprised anti-parallel β-sheet from β1to β3, and the Nudix domain was α + β fold, with mixed β-sheet of β4 to β9 surrounded by three α-helices of α1 to α3 (Fig. 1B). N-terminal residues (1-10) and residues (124-130) in loop L4 were disordered, but the presence of ADPR and metal resulted in order in loop L4. The Nudix motif (residues 67-89) folded into a loop-helix structure, not a loop-helix-loop structure, stabilized by an electrostatic network formed by salt links between Arg-81 and three Glu residues, Glu-73, Glu-82, and Glu-85. In general, thermophile proteins tend to have a short loop length, because flexibility of the loop reduces stability. Smaller loops, such as the loop-helix structure described above, are advantageous to TtADPRase. The TtADPRase structure of Gd3+ ternary complex was quite similar to those of the EcADPRase and MtADPRase (10Gabelli S.B. Bianchet M.A. Ohnishi Y. Ichikawa Y. Bessman M.J. Amezel L.M. Biochemistry. 2002; 41: 9279-9285Crossref PubMed Scopus (62) Google Scholar, 11Kang L.-W. Gabelli S.B. Cunningham J.E. O'Handley S.F. Amzel L.M. Structure. 2003; 11: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), although the detailed structures differed. N-terminal domain β-sheet was shorter, loop L4 was moved outward, and loop L5 tip was situated upward by 4 Å. TtADPRase has other properties that might contribute to stability, including a lower content of chemically unstable amino acids such as Asn, Cys, and Met (25Vieille C. Burdette D.S. Zeikus J.G. Biotechnol. Annu. Rev. 1996; 2: 1-83Crossref PubMed Scopus (150) Google Scholar), and a smaller surface area in the N-terminal domain than for EcADPRase and MtADPRase. The TtADPRase homodimer was stabilized by interactions in three regions. The first region contained an interface created between the N-terminal domains of both subunits, where the side chain of Thr-15 and the amide nitrogens of Arg-18 and Ile-19 made hydrogen bonds with the side chain of Glu-29 of another subunit. The second region contained an interface created between the N-terminal domain of one subunit and the Nudix domain of another subunit, where His-33 made a stacking interaction with Phe-105 of another subunit. The third region contained an interface created between the Nudix domains of both subunits, where Phe-97 made a stacking interaction with His-145 of another subunit, Ser-102 made a hydrogen bond with Glu-108 of another subunit, and Lys-109 made a salt bridge with Glu-151 of another subunit. The stereo structure and the scheme of hydrogen-bonding and stacking interaction between TtADPRase and ADPR are shown in Fig. 2 (A and B). The activity of TtADPRase was dependent on the presence of metal ions and was inhibited by acidic pH. ADPR was not hydrolyzed when crystals were prepared in solutions without metal ions for the binary complex with ADPR or prepared in an acidic pH for the ternary complex with ADPR and Zn2+ or Gd3+ (Table II). The binary complex without met" @default.
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• W2034304005 title "Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal" @default.
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