Article pubs.acs.org/biochemistry
ADP-Ribose Pyrophosphatase Reaction in Crystalline State Conducted by Consecutive Binding of Two Manganese(II) Ions as Cofactors Yoshihiko Furuike,† Yuka Akita,† Ikuko Miyahara,†,‡ and Nobuo Kamiya*,†,‡ †
Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan
‡
S Supporting Information *
ABSTRACT: Adenosine diphosphate ribose pyrophosphatase (ADPRase), a member of the Nudix family proteins, catalyzes the metal-induced and concerted general acid−base hydrolysis of ADP ribose (ADPR) into AMP and ribose-5′-phosphate (R5P). The ADPRhydrolysis reaction of ADPRase from Thermus thermophilus HB8 (TtADPRase) requires divalent metal cations such as Mn2+, Zn2+, or Mg2+ as cofactors. Here, we report the reaction pathway observed in the catalytic center of TtADPRase, based on cryo-trapping X-ray crystallography at atomic resolutions around 1.0 Å using Mn2+ as the reaction trigger, which was soaked into TtADPRase-ADPR binary complex crystals. Integrating 11 structures along the reaction timeline, five reaction states of TtADPRase were assigned, which were ADPRase alone (E), the ADPRase-ADPR binary complex (ES), two ADPRase-ADPR-Mn2+ reaction intermediates (ESM, ESMM), and the postreaction state (E′). Two Mn2+ ions were inserted consecutively into the catalytic center of the ES-state and ligated by Glu86 and Glu82, which are highly conserved among the Nudix family, in the ESM- and ESMM-states. The ADPR-hydrolysis reaction was characterized by electrostatic, proximity, and orientation effects, and by preferential binding for the transition state. A new reaction mechanism is proposed, which differs from previous ones suggested from structure analyses with nonhydrolyzable substrate analogues or point-mutated ADPRases.
E
and ADPR itself works as a second messenger in the calcium/ sodium signaling pathway.11 For these physiological phenomena, it is valuable to understand how ADPRases convert ADPR into totally innocent products. The metal-induced and concerted general acid−base catalysis in ADPRases generates a nucleophilic hydroxide ion from one water molecule in the catalytic center. The deprotonation of the nucleophilic water molecule followed by the attack of resultant hydroxide ion on the phosphorus atom of pyrophosphate is the rate-limiting step of ADPR hydrolysis, which is particularly important when we consider the reaction mechanism. The reaction mechanisms of ADPRases that have been proposed before are as follows: (i) The α-phosphate of ADPR undergoes nucleophilic attack by the hydroxide ion produced by a general base as indicated in Figure 1B.2 (ii) A nucleophilic water molecule is introduced into the catalytic center, sandwiched, and activated by the divalent metal-ion cofactors combined with ADPR pyrophosphate moieties. The structures of the reaction intermediates have been revealed using the nonhydrolyzable substrate analogue AMPCPR. (iii) Glutamate residues on a flexible loop surrounding the outside of ADPRase extract a proton from the nucleophilic water molecule as a
nzymes change their structures and chemical properties by interacting with substrates and cofactors during catalytic reactions. ADP-ribose pyrophosphatases (ADPRases), members of the Nudix family proteins (NFP), have been investigated by kinetic studies and X-ray crystal structure analyses, but the reaction mechanism of ADPRases is still under discussion.1−7 Along with other members of the NFP, the highly conserved Nudix motif GX5EX7REUXEEXGU (U, bulky aliphatic residues such as Ile, Leu, and Val; X, any amino acid residue) is preserved in ADPRases, beside the ADP-ribose binding site as represented in Figure 1A in addition to the binding site of ADP ribose (ADPR).8 An α-helix of the Nudix motif provides metal binding sites for divalent metal cations such as Mn2+, Zn2+, or Mg2+, which are essential cofactors (Figure 1A). ADPRases hydrolyze the pyrophosphate moiety of ADPR and produce AMP and ribose-5′-phosphate (R5P) as shown in Figure 1B. ADPR as a derivative of NAD+ in metabolic pathways is toxic at excessively high concentrations and functions in posttranslational modifications known as ADP ribosylations and in calcium/sodium signaling.9−11 ADP ribosylations are ADPR additive reactions toward Arg, Cys, and Asn residues in proteins that induce various physiological effects.9 When the concentration of ADPR is excessively high, ADP ribosylation leads to nonenzymatic polymerization of ADPR, and the polymers affect protein functions in a nonspecific manner.10 Some channel proteins include a Nudix domain to hydrolyze ADPR, © 2016 American Chemical Society
Received: August 9, 2015 Revised: February 2, 2016 Published: March 16, 2016 1801
DOI: 10.1021/acs.biochem.5b00886 Biochemistry 2016, 55, 1801−1812
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EXPERIMENTAL PROCEDURES
Overexpression and Purification of TtADPRase. Overexpression and purification procedures of TtADPRase were slightly modified from those previously reported.13,14 E. coli cells, BL21(DE3), were grown at 310 K on Luria-Bertaini culture plates. Overnight preculture from a single colony at 310 K was inoculated to main cultures for 7 h. Precipitated cells were rediluted in 20 mM Tris-HCl buffer solution including 50 mM NaCl at pH 8.0. After a heat treatment at 343 K for 10 min,15 ammonium sulfate was added at room temperature up to 1.35 M. TtADPRase in the supernatant was applied at 277 K into a hydrophobic column, Resource ISO (GE Healthcare BioScience Corporation), and separated by a linear gradient from 1.35 to 0.5 M ammonium sulfate in 1.5 M Na phosphate buffer at pH 7.0. Each fraction was analyzed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), and two or three fractions were strictly selected for subsequent procedures. After overnight dialysis to 50 mM Tris-HCl buffer solution at pH 8.0, anion exchange chromatography with a Resource Q column (GE Healthcare Bio-Science Corporation) was performed at 277 K. TtADPRase fractions eluted as a sharp peak in a linear gradient from 0 to 0.5 M NaCl were analyzed again by SDS−PAGE, and final fractions were selected for crystallization. While the previous procedures were performed at room temperature, most procedures in this study were carried out at 277 K. Crystallization of TtADPRase. TtADPRase crystals were grown by hanging-drop vapor-diffusion. The precipitant solution contained 0.24 M acetate buffer at pH 4.6, 0.32 M ammonium sulfate, 30% (w/v) glycerol, and 8−10% (w/v) polyethylene glycol (PEG) 20000. A sample solution with 20 mM Tris-HCl at pH 8.0 and 0.15 M NaCl was prepared to contain TtADPRase at a concentration of 4 mg/mL. The sample solution and the precipitant solution were mixed at a ratio of 1:1, and crystallization droplets of 8 μL were equilibrated against 300 μL of precipitant solution in a reservoir at 298 K. Three or 4 weeks were required to grow crystals up to 0.75 × 0.35 × 0.2 mm3. Preparation of TtADPRase-ADPR Binary Complex Crystals. The TtADPRase crystals in the apo-form were soaked overnight at 298 K into 5 mM ADPR solution in 0.24 M acetate buffer at pH 4.6, including 0.1 M Tris-HCl, 0.32 M ammonium sulfate, 30% (w/v) glycerol, and 10% (w/v) PEG 20000. The crystals resulted in enzyme−substrate (ES) complexes, but the ADPR hydrolysis reaction of TtADPRase could not start without divalent metal ions working as cofactors. Preparation of Reaction Intermediate Crystals after Mn2+ Ion Soaking. Mn2+ ions were soaked into TtADPRaseADPR binary complex crystals in a solution of 15 mM MnCl2 in 0.24 M acetate buffer at pH 4.6, including 5 mM ADPR, 0.1 M Tris-HCl, 0.32 M ammonium sulfate, 30% (w/v) glycerol, and 10% (w/v) PEG 20000 at room temperature. The crystals were held for 3, 6, 10, 15, 20, 30, and 50 min, and the reaction in each crystal was stopped by the quick exposure of the crystal into a nitrogen gas stream at 100 K (cryo-trapping technique). X-ray Diffraction Intensity Measurement. All data sets were collected at beamlines BL38B1 or BL44XU in SPring-8 (Japan). To obtain one data set from one crystal, two X-ray diffraction experiments were carried out at short wavelengths of 0.7−0.9 Å at a cryo-temperature of 100 K. One was a long exposure of X-rays to collect diffraction intensities at a higher
Figure 1. ADP-ribose pyrophosphatase from Thermus thermophilus; HB8 and ADP-ribose hydrolysis reaction. (A) Overall structure of TtADPRase in a homodimeric form. The Nudix motif (residues 67− 89) is colored in yellow. The flexible loops surrounding the outside of TtADPRase are indicated with dashed lines. (B) Chemical formulas of ADPR, AMP, and R5P. The divalent metal ion is indicated by M2+. The concerted general base and acid catalyses are shown. (C) Five reaction intermediates assigned along the reaction timeline. Relationships between the reaction states and the soaking times are also depicted.
general base. These proposals are all based on X-ray crystallography using AMPCPR or mutant ADPRases.2−4 Kinetic studies using point-mutated ADPRases have suggested that the highly conserved glutamate residues in the Nudix motif only provide binding sites for the metal ions but are not involved in the water activation process.6 In order to identify the reaction intermediates directly, we applied techniques of cryo-trapping X-ray crystallography. It is well-known that chemical reactions proceed even in a crystalline state. For example, a new reaction mechanism of DNA polymerase has been recently proposed based on structures of the reaction intermediates fixed by cryo-trapping techniques after the diffusion of metal-ion cofactors into the crystals as a reaction trigger.12 The soaking time of metal ions and small molecules into protein crystals of submillimeter size is very fast, usually less than 1 min. When time constants of crystalline-state reactions are longer than tens of minutes, the trajectories of the reactions can be evaluated by individual determinations of crystal structures along the timelines. The crystalline-state reaction in each of the crystals is stopped by the cryo-trapping technique, and X-ray diffraction intensities are measured from each crystal using monochromatic synchrotron radiation to collect a high quality data set. High resolution around 1.0 Å enables us to distinguish electron density distributions consisting of different reaction states. In this study, new investigations of dynamic structural changes and fine structural information uncovered the details of the ADPRase reaction mechanism, which turns out to be different from those previously proposed.1−7 Furthermore, we discuss our findings as related to metal-ion catalysis (the transition state preferential binding effect, the orientation effect, the proximity effect, and the activation of the nucleophilic water molecule), and general base catalysis (the orientation effect and the proton transfer). 1802
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+E′) was calculated as Frac(E+E′) = 1 − Occ(ADRP or ADPR*) for all points of the timeline. Frac(ES) at 0 or 3 min was equal to Occ(ADPR). Frac(ESM) at 6 min was calculated as Frac(ESM) = Occ(ADPR*). For the time points from 10 to 20 min, Frac(ESM) and Frac(ESMM) were calculated as Frac(ESM) = 1 − Occ(Glu82-A) and Frac(ESMM) = Occ(ADPR*) + Occ(Glu82-A) − 1, respectively, because Frac(E+E′) + Occ(ADPR*) = 1, Frac(ESM) = Occ(ADPR*) − Frac(ESMM), and Frac(ESMM) = Occ(Glu82-A) − Frac(E +E′).
resolution range around 1.0 Å. The other was a short X-ray exposure to compensate for the lack of reflections at a lower resolution range. After integrating diffraction intensities with HKL2000,16 the data obtained from the two experiments were merged. Reflections were truncated at a resolution where ⟨I⟩/ ⟨σ(I)⟩ was 2.0, and completeness was 95%. Furthermore, to confirm Mn2+ ions in the reaction intermediate crystals, an anomalous dispersion data set was obtained from each of the same crystals at an X-ray wavelength of 1.8 Å. Crystal Structure Analysis. Initial structures were obtained by the molecular replacement (MR) method using MOLREP in the CCP4 program suite.17 The search model used in this study was a structure of Zn2+ ion-soaked TtADPRaseADPR binary complex crystal, which was determined before this work (not submitted to PDB) with the MR method using the crystal structure previously reported5 (PDB-ID: 1V8R) as the search model. Restrained least-squares refinement was started from a resolution of 2.0 Å to the highest resolution using REFMAC5.18 At initial stages of refinement, temperature factors were treated as isotropic and shifted to be anisotropic at resolutions higher than 1.3 Å. When clear positive electron density distributions were found on the 2|Fo| − |Fc| maps at a 1.0 sigma level and |Fo| − |Fc| difference Fourier maps at a 3.0 sigma level, structure models were constructed on COOT.19 The anomalous difference Fourier maps were used to locate the Mn2+ ions. After restrained least-squares refinements using REFMAC5 at the highest resolutions, we shifted to SHELXL.20 Because the electron density maps obtained by SHELXL were slightly different from the ones obtained by REFMAC5, assignments of water molecules were checked again using the 2|Fo| − |Fc| maps at a 1.5 sigma level and the |Fo| − |Fc| difference Fourier maps at a 3.0 sigma level. In order to minimize the model biases, the structure models were verified by comparing the 2|Fo| − |Fc| maps with the composite omit maps generated by CNS21 at several stages of refinement. Finally, the 2|Fo| − |Fc| maps and the composite omit maps became almost identical (see Figure 3 and Figure S1). Chemical species around the catalytic site (ADPR, ADPR*, Glu82 in A-form, M1, and M2; see Results and Discussion) were assigned with occupancies less than unity in the restrained least-squares refinement. The occupancy values were changed step-by-step manually, and the peak heights of |Fo| − |Fc| difference Fourier maps were minimized to be less than the 3.0 sigma level in the final models. Hydrogen bond geometries in the structure model were carefully checked on a criterion for distance, 2.6−3.2 Å, and on for angle, 108−120°, based on the sp3 and sp2 hybrid-orbital configurations of the oxygen atom. Graphic representation of structures was performed using PyMol.22 Evaluation of Fractional Composition for Reaction States. Fractional compositions (Frac) of reaction states (E +E′, ES, ESM, and ESMM; see Results and Discussion) were evaluated using occupancy values (Occ) of chemical species around the catalytic site (ADPR, ADPR*, and Glu82 in Aform). The apo-form crystals were obtained from TtADPRase alone (E-state) in the absence of ADPR and divalent metal cations. The structure of the TtADPRase-ADPR binary complex corresponded to the ES-state at a reaction time of 0 min. Crystal structures obtained along the Mn2+ soaking timeline from 3 to 50 min contained several reaction states from ES to E′. The initial E-state and the final E′-state after the reaction could not be distinguished, and their fractional compositions were summed up as for the E+E′ state. Frac(E
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RESULTS AND DISCUSSION Crystals of Apo-Form TtADPRase and Its ADPR Binary Complex. Two ADPRases, Ndx2 and Ndx4, have been identified in T. thermophilus HB8.7 Ndx4 hydrolyzes ADPR specifically, whereas Ndx2 operates on not only ADPR but also flavin adenine dinucleotide. Thus, we focused on Ndx4 (TtADPRase) to elucidate the mechanism of ADPR hydrolysis. Crystallization conditions of TtADPRase in the apo-form have been reported previously.13 The crystals belong to the trigonal system, the space group is P3221, and the cell dimensions are a = 50 Å and c = 119 Å. Using these crystals, several structures have been analyzed at the highest resolution of 1.5 Å.5−7 We considered that this resolution might not be sufficient to resolve disordered structures anticipated in our time-resolved or cryotrapping X-ray crystallography. Thus, we first tried to obtain much higher resolution by further optimization of crystallization conditions and finally succeeded in improving the resolution up to 0.91 Å for apo-form TtADPRase crystals with the same space group and cell dimensions as in previous studies. A combination of 10% (w/v) PEG20000 and 30% (w/ v) glycerol instead of 18% (w/v) PEG4000 and 20% (w/v) glycerol used previously was effective for resolution improvement. The pH value of acetate buffer was decreased intentionally from 5.0 to 4.6 to reduce the hydration velocity of TtADPRase in crystals, as discussed later. The original samples were 11 apo-form crystals indicated as the E-state in Figure 1C. Nine of the 11 crystals were soaked overnight at 298 K in a solution including 5 mM ADPR, resulting in stable TtADPRase-ADPR binary complexes (ESstate), in which the ADPR hydrolysis reaction did not proceed because of the absence of divalent metal cations. The ADPR concentration of 5 mM was carefully selected because ES-state crystals prepared at much higher concentrations of ADPR, for example, 15 mM, were seriously damaged during the following Mn2+ ion soaking. The stable ES-state crystals had occupancies of ADPR around 0.8, and the remaining 0.2 was assignable as water molecules, as shown in Figure 2 (see also Figure 3 and Figure S1). Statistics on diffraction intensity measurements and structure refinements are summarized in Table S1. Timeline for Cryo-Trapping Experiments. Seven of the nine ES-state crystals were further soaked in a solution including 15 mM MnCl2 at pH 4.6, then the ADPR hydrolysis reaction was started in the crystalline state (Figure 1C). The total hydrolysis reaction from ADPR to AMP and R5P, composed of several elementary reactions, proceeded in one direction as consecutive reactions (see Figures 3 and 4). The forward velocities for the elementary reactions should be much higher than the backward ones because only reactants existed at the initial stages for all elementary reactions. The reaction was stopped at various periods from the starting time of Mn2+ ion soaking by putting the crystals quickly into a nitrogen gas stream at 100 K. X-ray diffraction experiments were performed 1803
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ates (ES-, ESM-, and ESMM-states) were evaluated. Although all eight structures from 0 min (ES-state) to 50 min include the original E-state of 20−30%, the structures of the ESM-state at 6 min and the ESMM-state at 20 min are sufficiently assignable as intermediate states. Hereafter, structural information obtained from the crystal structures in this study is described in order to explain the reaction mechanism of TtADPRase, composed of the five states, E, ES, ESM, ESMM, and E′. Outline of Events Proceeding in the Reaction Cavity of TtADPRase. ADPR is composed of three moieties: adenosine, a central pyrophosphate, and a terminal ribose. The β-phosphate (PB) of the central pyrophosphate moiety, at the terminal ribose side, is recognized by two parallel hydrogen bonds of Arg54 as shown in Figure 5A. The α-phosphate (PA) at the adenosine side, which forms a hydrogen bond with the main-chain nitrogen atom of Leu68, was closer to the Nudix motif than PB. Two alternative conformations were found for the highly conserved Glu82, in the A- and B-forms. To recognize the conservation of amino acid residues, multiple sequence alignments of ADPRases are given in Figure S3. The A-form of Glu82 is exposed to the hydrophilic region in the reaction cavity and stabilized by the highly conserved Arg81, whereas the B-form is buried in the hydrophobic region (Figure 5B). The A-form of Glu82 in the E-state is pushed into the Bform by steric hindrance and charge repulsion of PA in the ESand ESM-states (Figure 4). The highly conserved Glu85, of previously unknown function, is proximate to the A-form of Glu82 and to Arg81. The conserved Glu73 also forms hydrogen bonds to Arg81 supported by Glu85 and the A-form of Glu82. Glu86 fixed by the structurally conserved Ala66 is the only residue located near both PA and PB of ADPR among the highly conserved residues in the α-helix of the Nudix motif. The octahedral coordination sphere of the Mn2+ ion (M1) is observed beside Glu86 in the ESM-state (Figure 6A). The six ligands are Glu86, PA, PB, the carbonyl group of Ala66, and two water molecules (W1 and W2), which are supported by the structurally conserved Gln52. Along with the M1 binding, PA is distorted into a conformation of a pyrophosphate moiety in ADPR*. PA rotates roughly 60° around a chemical bond between PA and the central oxygen atom of the pyrophosphate moiety, and this conformation is suitable to bind the second Mn2+ ion (M2) found in the ESMM-state (Figure 6B). Furthermore, the population of Glu82 in the A-form increases to bind M2 (Figure 4). The observed ligands of M2 are PA in ADPR*, the A-form of Glu82, and only one water molecule (W3) that always forms a hydrogen bond to Glu85. Between the reaction time points of 20 and 30 min, the products AMP and R5P, as well as two metal ions, M1 and M2, are released from the reaction cavity, resulting in the empty cavity of the E′state (Figure 3). This means that the nucleophilic water molecule should be activated within the coordination sphere of M2 in the ESMM-state as a climax of the TtADPRase reaction. Consecutive Binding of Two Mn2+ Ions. The pyrophosphate moiety of ADPR changed its conformation at PA, resulting in ADPR*. PA is recognized from one side by hydrogen bonds from Leu68 and a water molecule (W4 in Figure 6B), and is allowed to move near M1. When M1 binds, the PA oxygen atom rotates to be closer to that of PB. Because ADPR* is generated before the ESMM-state, the new conformation of ADPR* may be important as a precursor in the pyrophosphatase reaction. Similar proximities of two phosphate groups are frequently found in other ADPRaserelated enzymes.12,23,24 Until M1 binds, Glu86 has two
Figure 2. ADPR structure in the TtADPRase-ADPR binary complex crystal. ADPR with an occupancy of 0.8 and the remaining water molecules (red spheres, occupancies 0.2) were refined in A, and only the ADPR molecule with an occupancy of 0.8 was used in the calculations of electron density maps in B. The same water molecules in A were drawn for comparison. 2|Fo| − |Fc| electron density maps (gray) and |Fo| − |Fc| difference Fourier maps (green) were drawn at 1.0- and 3.0-sigma levels, respectively.
at leisure because the reactions in the crystals were completely stopped at this cryo-temperature. The reactions were terminated by cryo-trapping techniques at 3, 6, 10, 15, 20, 30, and 50 min. These periods from the start of the reaction and the Mn2+ ion concentration were based on various preliminary experiments by trial and error. Because Mn2+ ions work as a reaction trigger, the soaking velocity into the ES-state crystals should be fast for the low heterogeneity required in each of the time-resolved structures. A high concentration is suitable to increase the soaking velocity, but an extremely high concentration affects ES-state crystals as mentioned above. While selecting 15 mM as an optimum, we found that two Mn2+ ions (M1 and M2 in Figure 3) were introduced consecutively into the reaction cavity, and two intermediate states (ESM- and ESMM-states) were realized. Time points of 3 and 6 min for cryo-trapping were used to catch the structure of the ESM-state. The pH value of Mn2+ ion soaking solution was maintained at 4.6 to slow down the hydration velocity of TtADPRase in the crystals. Time points of 10, 15, and 20 min were used to identify the ESMM-state, and 30 and 50 min were used to confirm that the crystalline-state reaction was completed at the E′-state where the structures around the reaction cavity were recycled to the initial E-state. Assignment of Intermediate States in the TtADPRase Reaction. Along the reaction timeline shown in Figure 1C, we observed successive insertions of two Mn2+ ions into the crevice between the Nudix motif and ADPR at 3−20 min, as shown in Figure 3. The existence of Mn2+ ions was clearly confirmed from anomalous difference Fourier maps (Figure S2). The electron density distributions of ADPR became faint at a reaction time point of 30 min as shown in Figure 3H, and the structure around the reaction cavity in the E-state was restored again at 30 and 50 min (E′-state). The resolutions of all data sets were around 1.0 Å; the highest resolution was 0.91 Å, and the lowest 1.22 Å, as listed in Table S1. The high resolution enables us to determine partial occupancies of chemical species in the crystals and to distinguish alternative conformers, as shown in Figure 4. Occupancies are highly correlated to temperature factors, and the reliabilities of occupancies are low enough that errors may be 0.1−0.15 in this study. The reliabilities expected from the restrained least-squares refinement are in the order of ADPR and ADPR* (described later) > Glu82 > Mn2+. Using the occupancies of ADPR, ADPR*, and one conformer of Glu82 (Glu82-A), fractional compositions for the reaction intermedi1804
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Figure 3. continued
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Figure 3. continued
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Figure 3. Stereodrawings of structure models obtained in cryo-trapping crystallography of TtADPRase. 2|Fo| − |Fc| electron density maps are drawn at a 1.0-sigma level.
hydrogen bonds toward Ala66. Because one of two hydrogen bonds is generated between the carboxylate oxygen atom of
Glu86 and the carbonyl group of Ala66, Glu86 should be protonated in E- and ES-states. From the ESM-state, M1 1807
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Figure 4. Occupancy of chemical species and fractional composition of reaction states. Occupancies of chemical species (ADPR, ADPR*, Glu82 in A-form, M1, and M2; see text.) and fractional compositions of reaction states assigned are listed in the upper table. Fractional compositions assigned from occupancies of ADPR and ADPR* are colored in red. Fractional compositions for ESM# and ESMM$ depicted in blue were calculated using occupancies of ADPR* and Glu82 in A-form. All of the fractional compositions assigned are also graphically indicated along the reaction timeline with the estimated error values in the lower part. E+E′, ES, ESM, and ESMM are depicted in blue, red, yellow, and green, respectively.
Figure 5. Stereodrawings of the structure model and interactions at the catalytic sites. (A) The positional relationships and interactions among the catalytic species. The yellow model indicates the Nudix motif, and the white model includes structurally conserved amino acid residues from Gln52 to Ala66. (B) Two conformations of Glu82 observed in the ESM-state. 2|Fo| − |Fc| electron density distributions are drawn at a 1.0 sigma level.
interrupts the hydrogen bond, and Glu86 may be deprotonated. M1 binding to Glu86 and the pyrophosphate moiety of ADPR
enables PA to prepare the binding site for M2. There are only three observed ligands for M2: PA, the A-form of Glu82, and 1808
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nucleophiles because of the long distances and unfavorable directions toward PA. On the basis of the positional relationships of PA, Glu82, and W3, we assume here the existence of octahedral or trigonalbipyramidal coordination spheres for M2. Octahedral structures are frequently found, and trigonal-bipyramidal complexes of Mn2+ are occasionally observed.26 In addition, the ligation of a hydroxide ion can induce a structural change from octahedral to trigonal-bipyramidal.27 In each case, if a water molecule (Wn) were positioned to complete the coordination sphere, it would be close to ligate to M2 within hydrogen bonding distances to Glu82, W3, and W4, as shown in Figure 6B. The hypothetical Wn should be located at one of the vacant coordination sites in the octahedral or trigonal-bipyramidal structures. The position of Wn is close to PA and thus suitable to attack the PA phosphorus atom in an in-line manner.2−4 Thus, we propose that M2, stabilized by PA, Glu82, and W3, provides a coordination site for the nucleophilic water Wn. M2 polarizes the nucleophilic water molecule to deprotonate it as a Lewis acid,27 and the resulting hydroxide ion attacks PA to hydrolyze ADPR into AMP and R5P. General Base. In previous reports, 2−4 Glu166 in HmADPRase and Glu142 in MtADPRase were proposed to be the general base. In the HmADPRase structure fixed by the nonhydrolyzable substrate analogue AMPCPR, the glutamate residue in the flexible loop (see Figure 1A), surrounding the outside of ADPRases, plunges into the catalytic site and forms hydrogen bonds to the nucleophilic water molecule to work as a general base. TtADPRase has Glu127, Asp128, and Glu129 in the corresponding flexible loop. In our cryo-trapping structures, however, the flexible loop was not fixed around the catalytic site, and the orientations of their side chains could not be assigned during the whole reaction (Figure 7). The
Figure 6. Binding sites M1 and M2 for two Mn2+ ions. (A) Structural change of ADPR by the ligation of M1. (B) The structure around M2. 2|Fo| − |Fc| electron density distributions are drawn at a 1.5 sigma level. The bond lengths are depicted in angstrom units.
one water molecule (W3). The M2 binding site may be more fragile than the M1 site. While the occupancy of M2 increased from 0 to 0.3 (Figure 4), the population of Glu82 in the A-form increased to 0.8. In general, occupancies and temperature factors of atoms are strongly correlated in protein crystallography even at high resolutions around 1.0 Å. If the temperature factors of the M2 site were much higher than those assigned, the corresponding occupancies would be much larger than the value of 0.3 in the ESMM-state at a reaction time of 20 min. Nonetheless, the parallel increments of M2 and Glu82 occupancies in the reaction timeline indicated that M2 pulled out Glu82 from the B-form into the A-form. The B-form of Glu82 should be protonated and neutral in order to be stabilized in the hydrophobic environment. In contrast, the A-form must be deprotonated and negatively charged to interact with M2. Including the flipping movement of Glu82, the electrostatic charge can be balanced around the metal ions in the TtADPRase reaction cavity. The increase of positive charges is +2 for M1 and +2 for M2 for a total of +4. The increase of negative charges is −2 for ADPR, −1 for Glu86, and −1 for the Glu82 flipping movement, for a total of −4. The complete balance of electrostatic charges is of great importance in determining the mechanisms of enzymatic reactions. Specifically, M1 is attracted by three negative charges, from PA, PB, and Glu86, while M2 is fixed by two negative charges, from PA and Glu82. PA is thus shared by both M1 and M2. The imbalance of negative charges around M1 and M2 may result in the stronger Lewis acidity of M2 compared to M1.25 M2 is a better candidate for activating a nucleophilic water molecule. Furthermore, the position of M1 is not appropriate in the in-line mechanism,2−4 and the water molecules ligated to M1 (W1 and W2 in Figure 6A) cannot possibly act as
Figure 7. 2|Fo| − |Fc| electron density maps around the flexible loop region constructed at the 1.0-sigma level in the ESMM-state. Residues from 124 to 128 were not assignable. One Mn2+ ion was confirmed by |Fo| − |Fc| anomalous difference maps at 3.0-sigma, but the location is different from the third metal binding site reported in previous studies.2−4
deprotonation of water molecules does not usually occur within the coordination sphere of Mn2+, which is known as Mn[H2O]62+, even at neutral pH.28 Because the ADPR hydrolysis reaction occurred in the TtADPRase crystal at pH 4.6, a negatively charged general base should exist in our cryotrapping crystal structures, except in the flexible loop region. Other authors2−4 have also proposed the participation of a third metal ion (M3) in ADPR hydrolysis, which binds the 1809
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Figure 8. Reaction mechanism proposed in general for ADPRases. Amino acid residues in the Nudix motif are shown in yellow. The structurally conserved residues are colored in blue. These color indications correspond to Figure S3. ADPR and water molecules are drawn in green and red, respectively.
substrate (ADPR) and cofactor (Mn2+ ions) to the enzyme should be discussed. In this study, the binding sequence was designed as ADPR first (ADPRase-ADPR binary complex, ESstate) and Mn2+ ions second (ADPRase-ADPR-Mn2+ reaction intermediates, ESM- and ESMM-states) in order to satisfy an experimental requirement of cryo-trapping crystallography that the diffusion rate of reaction trigger into crystals should be as fast as possible. In fact, a soaking time of 5 mM ADPR into the apo-form ADPRase crystal was set as overnight, and that of 15 mM Mn2+ solution was in the order of 1 min. Because of the lack of knowledge on the real binding sequence, ADPR first and Mn2+ second were our assumptions at the beginning of this work. After experiments, the following two points were recognized: (i) Mn2+ ions were not observed in the reaction cavity in E′-state (Figure 3), although the Mn2+ ion concentration was 15 mM in the reaction medium, and (ii) there were many hydrogen bonds as well as electrostatic interactions between ADPR and TtADPRase (Figure 5A). These results may relate to the stable binding of ADPR to ADPRase and unstable binding of Mn2+ ions without ADPR, and are consistent with our assumption. Consistent with a previous report,5 we confirmed that Glu86 and Glu82 construct the metal binding sites. The clear elimination of activity by point-mutations of Glu86 and Glu82 demonstrates that the metal ions are important in the ADPRase reaction.6 The M1 binding stabilized by the deprotonated Glu86 transforms ADPR to ADPR* by a preferential binding effect30 accompanying the common distortion of pyrophosphates. M1 exhibits an orientation effect30 to prepare the binding site for M2. M2, stabilized by PA, Glu82, and W3, achieves a proximity effect30 by inviting the nucleophilic water molecule into the Wn site, which is in the best position to attack PA in an in-line manner. In addition to the positional arrangement, Wn may be assisted by M2 to be polarized, and the proton may be extracted from Wn by the general base of Glu82 in cooperation with Glu85 and water molecules. On the basis of these observations, the new reaction mechanism of TtADPRase is proposed here. The enzyme alone is represented in A of Figure 8. Accompanying the
nucleophilic water molecule in a sandwich manner with M2. These proposals are also based on static crystal structures using AMPCPR. However, our cryo-trapping crystallography did not detect M3 (Figure 3). The absence of M3 was also confirmed in anomalous difference Fourier maps (Figure S2). Glu82 can possibly act as a general base in the water activation process of TtADPRase. One of the two carboxylate oxygens of Glu82 (OE1) accepts a bond from M2, whereas the other (OE2) is free and can thus extract a proton from Wn as the general base (Figure 6B). Monodentate interactions of carboxylates toward divalent metal cations are frequently observed in enzymes, and the internal proton transfer from the ligand water molecule to the carboxylate group ligated to the metal cations has already been predicted.29 Glu85 is a highly conserved residue as are Glu82 and Glu86 in the Nudix motif (Figure S3), but its function has not been revealed. We note that Glu85 can possibly form a proton transfer pathway from Wn to W5, which exists on the outside surface of TtADPRase (Figure 5A). The direction of the putative proton transfer pathway is opposite to the direction of nucleophilic attack by the hydroxide ion toward PA. This is effective to prevent the recombination of the nucleophilic hydroxide ion with the extracted proton into a water molecule. In contrast, Glu82 and Glu85 are not conserved in GDP mannose hydrolase (GDPMH), although GDPMH is classified as a NFP protein.8 GDPMH hydrolyzes GDP mannnose into GDP and mannose with only one metal ion, which corresponds to M1 in the case of ADPRases. The nucleophilic water molecule is activated by a His residue working as a general base within the binding sphere of M1.8 The reaction mechanism of GDPMH is totally different from that of ADPRases, and there is no water activation site around the amino acid residues corresponding to Glu82 and Glu85 of the Nudix motif. Therefore, there is no necessity to preserve Glu82 and Glu85 in GDPMH. This significant difference between GDPMH and ADPRase paradoxically implies the importance of Glu85 in the water activation process proceeding in the catalytic center along with Glu82. Reaction Mechanism of TtADPRase. Before describing the reaction mechanism of TtADPRase, the binding order of 1810
DOI: 10.1021/acs.biochem.5b00886 Biochemistry 2016, 55, 1801−1812
Article
Biochemistry Accession Codes
insertion of ADPR into the reaction cavity, one proton is accepted from outside to the deprotonated Glu82 (B). The first divalent metal cation (M1) is introduced into the space surrounded by the protonated Glu86, Ala66 that forms a hydrogen bond to the carboxylate oxygen atoms of Glu86, PA, and PB of ADPR (ADPR* after the M1 binding), and two water molecules (W1 and W2) supported by the structurally conserved Gln52. Until M1 binds, Glu86 should be protonated for the hydrogen bond toward Ala66. M1 induces Glu86 deprotonation, and the detached proton transfers to the outside (C). The second metal cation (M2) successively introduced is ligated by PA of ADPR*, Glu82 which is again deprotonated (the detached proton goes out), and the water molecule (W3) supported by the negatively charged Glu85 (D). M2 is also proximate to W4 supported by the hydrogen bond from the main chain of Leu68. Wn is deprotonated by the general base, Glu82, and the nucleophilic hydroxide ion generated attacks the phosphorus atom of PA, resulting in pyrophosphate cleavage in an in-line manner. The remaining PB captures one proton from structurally conserved Arg54 working as a general acid. The proton extracted from Wn might be transferred to Glu85 (E) and then to outside of the ADPRase via W5. Two products, AMP and R5P, and two metal cations, M1 and M2, are released from the reaction cavity accompanying two proton-captures, one to Arg54 via W6 and the other to Glu86. Then, the increase and decrease of protons and charges are satisfied (A), and the catalytic cycle is complete. The amino acid residues depicted in Figure 8 are all conserved as shown in Figure S3. The metal ions and the water molecules are also observed at the same positions, and the binding modes of ADPR are almost identical in TtADPRase_Ndx2, EcADPRase, MtADPRase, and HmADPRase. The mechanism including three metal ions, M1−M3, has been based on classic crystallography with a nonhydrolyzable substrate analogue, AMPCPR.2−4 In contrast, M3 was not detected in our cryo-trapping studies with the real substrate, ADPR. Furthermore, the fixation of the flexible loop and the sandwich structure of M2−water−M32−4 were not observed. The participation of M3 and the flexible loop in the ADPR hydrolysis reaction might be the results of evolution to realize much higher efficiency or much wider substrate diversity. If structural heterogeneity in the crystalline-state reactions is overcome, cryo-trapping crystallography can reflect different cutting faces of enzymology based on dynamic atomic structural information. The mechanism proposed here will be shared as minimal essentials of ADPRase reactions in the fields in which ADPRases and Nudix family proteins are involved.
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PDB accession codes for the structure analyses are as follows: 3X0I, 3X0J, 3X0K, 3X0L, 3X0M, 3X0N, 3X0O, 3X0P, 3X0Q, 3X0R, and 3X0S.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-6-6605-3131. E-mail:
[email protected]. Author Contributions
The manuscript was written by Y.F. and N.K. The highresolution crystals around 1.0 Å were initially obtained by Y.A. Funding
Funding was provided by JSPS KAKENHI Grants Nos. 21370049 and 13J05019. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the staff members at the beamlines BL38B1 and BL44XU of SPring-8, Japan (proposal numbers: 2001B1428, 2012B1203).
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ABBREVIATIONS AMP, adenosine-monophosphate; NAD, nicotinamide adenine dinucleotide REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b00886. Multiple alignments of ADPRases; statistics on diffraction intensity measurements and structure refinements; stereo-drawings of structure models obtained by cryotrapping crystallography of TtADPRase; anomalous difference Fourier maps; and multiple alignment of ADPRases (PDF) 1811
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