Tài liệu Báo cáo khoa học: Crystal structure of the cambialistic superoxide dismutase from Aeropyrum pernix K1 – insights into the enzyme mechanism and stability pdf

j= hkl i IIhkl;j , where Ihkl,j is the intensity of P P observation Ihkl,j and is the average of symmetry-related observations of a unique reflection c Rcryst = ||Fo| ) |Fc|| ⁄ |Fo|, where Fo d and Fc are observed and calculated structure factor amplitudes, respectively Rfree was calculated using a randomly-selected 5% of the dataset that was omitted from all stages of refinement eRamachandran plots were prepared for all residues other than Gly and Pro a 600 FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS T Nakamura et al Crystal structure of SOD from A pernix K1 A B 90° Fig Crystal structure of ApeSOD (A) Monomer structures of apo (green), Mn-bound (magenta) and Fe-bound (cyan) ApeSODs are superimposed and shown as a stereo view The metal cofactor of the Mn-bound form is indicated by a ball (B) Tetramer structures of ApeSOD viewed from two directions Chains A, B, C and D are shown in red, yellow, green and blue, respectively (C) ApeSOD (red) superimposed with the SOD of P shermanii (blue; Protein Data Bank code: 1AR5) [6] The structure of ApeSOD is viewed from the same directions as in (B) The Mn-bound form is shown as the representative of ApeSOD in (B) and (C) Prepared with PYMOL [44] C of the N-terminal extended region and the following two a-helices, and the globular, (a + b)-type C-terminal domain Oligomeric structure The A ⁄ B and C ⁄ D chains formed dimers in the crystal packing This dimerization buried 27% of the accessible surface area of each monomer These two dimers associated to form a tetramer in the asymmetric unit (Fig 1B) The tetramerization buried 13% of the accessible surface area of each dimer Neighboring tetramers came into loose contact with each other in the crystal packing Similar molecular arrangements have been found in the crystal structures of SODs from several sources, such as Sulfolobus solfataricus [18], Mycobacterium tuberculosis [19], Aquifex pyrophilus [20] and P shermanii [6] These SODs are assumed to be tetra- 90° meric in solution Figure 1C illustrates the superimposition of ApeSOD with the cambialistic SOD from P shermanii The overall structures of these enzymes were similar; the rmsd of the 747 Ca atoms was ˚ 0.695 A ApeSOD eluted from the gel-filtration column with the elution volume for a molecular mass of 57 kDa (Fig 2A) Because the calculated molecular mass of the ApeSOD monomer is 24 577 Da, the gel filtration results suggested a dimeric association; similar results have previously been reported for the same protein [16,21] A second gel filtration through a Superdex 200 column also suggested that ApeSOD has a dimeric structure in solution (data not shown) These findings are in contrast to the results obtained in the crystallographic study (described above), which demonstrated that ApeSOD has a tetrameric structure To determine whether ApeSOD polypeptide associations were FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 601 Crystal structure of SOD from A pernix K1 T Nakamura et al Active site geometry A 1009 A280 MW (kDa) 10 10 11 12 13 Elution volume (mL) B Residuals 15 10 20 25 Elution volume (mL) 0.05 0.00 –0.05 1.5 A280 1.0 0.5 0.0 6.85 6.90 6.95 7.00 7.05 7.10 Radius (cm) Fig Assembly of ApeSOD chains in solution (A) Representative gel filtration chromatogram of ApeSOD and calibration curve (inset) The standard proteins are albumin (1), ovalbumin (2), chymotrypsinogen A (3) and ribonuclease A (4) The estimated molecular mass of ApeSOD is 57.0 kDa (B) Sedimentation equilibrium distribution of ApeSOD The line reflects the best fit of the data and indicates that the apparent molecular mass is 96 265 Da The deviation between empirical data and the fitted line is plotted in the upper panel different in crystals and in solution, we used ultracentrifugation to accurately measure the molecular mass of ApeSOD in solution The result (96 265 Da) clearly showed tetrameric assembly of ApeSOD in solution (Fig 2B) Because ultracentrifugation is a direct, accurate measurement, we conclude that ApeSOD exists as a tetramer in solution Each monomer had an independent metal-binding site at the interface of the two domains, which consists of four side chains: two (His31 and His79) from the N-terminal domain and two (Asp165 and His169) from the C-terminal domain (Fig 3) In Mn-reconstituted ApeSOD, the metal ion was five-coordinate in trigonal bipyramidal geometry (Fig 3A) Three of the ligands, OD2 of Asp165, NE2 of His79 and NE2 of His169, formed an equatorial plane The other protein ligand, NE2 of His31, bound to the metal, in the company of a water oxygen, from the apical positions The manga˚ nese was only 0.06 A out of the equatorial plane (Table 3) The angles around the metal cofactor suggested that the ligation form of Mn in ApeSOD is trigonal bipyramidal In Fe-reconstituted ApeSOD, the metal was coordinated with six ligands: the five same ligands in the Mn-reconstituted enzyme and an additional water oxygen, which, together with the OD2 of Asp165, the NE2 of His79 and the NE2 of His169, formed an equatorial plane (Fig 3B) The metal ion and the addi˚ tional water oxygen were only 0.03 and 0.04 A, respectively, out of the equatorial plane defined by the other three atoms (Table 3) The angles around the metal ion indicated that Fe-bound ApeSOD contains distorted octahedral coordination around the metal cofactor The absence of an anomalous Fourier map demonstrated that the active site in apo-ApeSOD did not contain a metal cofactor (Fig 3C) However, the side chains and apical water coordinating the metal center had the same configurations as those in the metalbound ApeSOD This implies that the conformation around the active site of ApeSOD is independent of the presence of a metal cofactor Figure 3D shows the superimposition of the active site structures of apo, Mn-bound and Fe-bound ApeSODs The most significant difference among them ˚ was related to the OH of Tyr39, which shifted 1.1 A toward the apical water molecule upon Fe binding (Fig 3D) The shift upon Mn binding was negligible, and no significant differences were observed in other Fig Active site structure of ApeSOD Active site structures of Mn-bound (A), Fe-bound (B) and apo (C) ApeSODs (A–C) The orange map represents the rA-weighted Fo)Fc electron density map at the r level, where the indicated residues are excluded from the calculation of the structure factor The blue map represents the anomalous difference map contoured at 10 r Schematic representations of coordinated atoms are shown on the right in (A–C), where oxygen, nitrogen and metal atoms are represented by red, blue and black balls, respectively (C) The anomalous difference map was not seen even when the sigma level was set to (data not shown) (D) Apo (green), Mn-bound (magenta) and Fe-bound (cyan) structures are superimposed, and the residues around the active site are shown via stick models The red, magenta and cyan balls represent the active site water molecules in apo, Mn-bound and Fe-bound ApeSODs, respectively Balls labeled ‘Metal’ are Mn or Fe (E) Showing the superimposition of the active site structures of the Mn- (magenta) and Fe-bound (cyan) forms of P shermanii SOD Water molecules and metal atoms are shown in the same way as in (D) Prepared with PYMOL [44] 602 FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS T Nakamura et al Crystal structure of SOD from A pernix K1 A WAT D165 (OD2) H169 (NE2) H79 (NE2) H31 (NE2) B WAT H169 (NE2) D165 (OD2) H79 (NE2) WAT H31 (NE2) C WAT D165 (OD2) H169 (NE2) H79 (NE2) H31 (NE2) D E FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 603 Crystal structure of SOD from A pernix K1 T Nakamura et al Table Distances and angles around the metal ions For the identity of the atoms, see Fig Mn ˚ Distance (A) Metal – equatorial plane Wata – equatorial plane Angle (°) D165-M-H79 H79-M-H169 H79-M-wata Wata-M-H169 H169-M-D165 H31-M-D165 H31-M-watb a Fe 0.06 ± 0.01 0.03 ± 0.02 0.04 ± 0.03 104.8 ± 1.3 136.1 ± 0.8 97.2 151.5 77.1 74.5 111.1 82.1 169.5 118.8 ± 0.8 82.7 ± 0.6 170.1 ± 1.5 Water on the equatorial plane b A ± ± ± ± ± ± ± 2.9 2.1 1.9 0.6 1.5 1.2 1.5 B Water at the apical position Table Interactions in SODs ApSOD, SOD from A pyrophilus; SsoSOD, SOD from S solfataricus ApeSOD Number of intrasubunit ion pairs ⁄ monomer Number of intrasubunit ion pairs ⁄ residue Number of intersubunit ion pairs ⁄ tetramer Number of intersubunit ion pairs ⁄ residue SsoSOD 0.033 0.024 24 0.028 24 0.029 ApSOD TthSOD 14 0.066 22 0.026 0.030 0.0049 active site residues between Mn- and Fe-bound ApeSODs, although slight changes in His31, His79 and Asp165 were observed between the metal-bound and apo ApeSODs The shift of the conserved Tyr residue depending on the metal cofactor does not appear to be a common feature among cambialistic SODs because, in the case of the cambialistic SOD from P shermanii, no significant difference was found between the active sites of the Mn- and Fe-bound forms (Fig 3E) Stability in organic medium After we elucidated the tertiary structure of ApeSOD, we were able to compare the number of ion pairs among thermophilic SODs Table summarizes the data obtained from two species each of archaea and bacteria A single ApeSOD polypeptide was found to have seven intrasubunit ion pairs, whereas 24 intersubunit ion pairs were found in the ApeSOD tetrameric assembly This is significantly higher than the number of ion pairs found in the SOD from the thermophilic bacterium T thermophilus (TthSOD) [22] Because electrostatic interactions in proteins are more domi604 C Fig Thermal stability of ApeSOD and TthSOD Residual activities of ApeSOD (closed circles) and TthSOD (open circles) after incubation at 85 °C in the aqueous solution (A) and the solution containing 40% ethylene glycol (B) are plotted against incubation time (C) ApeSOD (closed symbols) and TthSOD (open symbols) were incubated for h at 60 °C (circles), 70 °C (squares) or 80 °C (triangles) in a solution containing various concentrations of ethanol The residual activities after the incubation are plotted against the ethanol concentration nant when the solvent polarity is lower, we hypothesized that ApeSOD would be more stable than TthSOD in an organic solvent When we compared the stabilities of ApeSOD and TthSOD, we found that the two enzymes were similar in aqueous solution at temperatures up to 85 °C (Fig 4A) However, when the solvent contained 40% ethylene glycol, TthSOD was inactivated by incubation FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS T Nakamura et al at 85 °C, whereas ApeSOD remained active (Fig 4B) Inclusion of ethanol in the solution destabilized both ApeSOD and TthSOD However, the extent of the destabilization was more significant in TthSOD than in ApeSOD (Fig 4C) In other words, ApeSOD was more stable than TthSOD under organic conditions This was not unexpected given the number of ion pairs in ApeSOD and TthSOD The larger number of electrostatic interactions in ApeSOD contributed to its stability in a low-polarity solvent Discussion In the present study, we crystallized ApeSOD and determined its tertiary structure The asymmetric unit contained four polypeptides that consisted of two homodimers Although both crystallographic study and analytical ultracentrifugation indicated that ApeSOD was a tetrameric protein, gel filtration suggested that the protein was dimeric (Fig 2A); in other words, the molecular mass estimated by gel filtration was lower than the true value Similar results have also been reported for other homologous SODs, such as those from S solfataricus [23] and P shermanii [24] Ursby et al [18] reported that the estimated molecular mass of the S solfataricus SOD increased when the column was recalibrated with thermophilic proteins from the same source In cases such as these, analytical ultracentrifugation, rather than gel filtration, is a powerful tool for accurately determining the oligomeric structure of SOD enzymes We observed five-coordinate and six-coordinate structures of metal ions in Mn-bound and Fe-bound ApeSOD crystal structures, respectively In the sixcoordinated Fe-bound ApeSOD, an additional water molecule was found in the equatorial plane (Fig 3B) Although trigonal bipyramidal 5-coordinate structures are often observed around bound metals of SODs, the octahedral six-coordinate crystal structures are detected only in exceptional cases; for example, in the cryo-trapped form of E coli Mn-SOD [25], in Fe-substituted Mn-SOD of E coli [3], in peroxide-soaked MnSOD of E coli [26] and in TthSOD complexed with azide, a SOD inhibitor [27] It remains unclear why the six-coordinated structure was observed in Fe-bound ApeSOD, but not in the Mn-bound form, because the crystal contained neither superoxide substrate, nor anionic inhibitors The answer to this question will shed light on the reaction mechanism of this cambialistic SOD Several enzymological properties known for ApeSOD [16] can be related to the structural characteristics in the active site Cambialistic SODs can be divided into two groups: one with almost the same Crystal structure of SOD from A pernix K1 activity in the Mn- and Fe-forms, and the other exhibiting low activity in the Fe-form and high activity in Mn-form ApeSOD belongs to the latter group [16] This was also confirmed by the activity assay, which demonstrated that ApeSOD was approximately 20-fold more active in its Mn-bound form than in its Fe-bound form (Table 1) We propose two possible explanations for this finding One is related to inhibition of the binding of the superoxide substrate and the other to product inhibition by hydrogen peroxide There are two mechanisms proposed for the reaction cycle of Mn ⁄ Fe-SOD: the 5-6-5 mechanism [27] and the associative displacement mechanism [28] In the former model, the metal is five-coordinated in the resting state and six-coordinated when the substrate superoxide is bound In the latter model, the association of superoxide is concomitant with displacement of one of the oxygen ligands In both mechanisms, the superoxide substrate coordinates to the metal center from the equatorial plane, and the coordination site is the same as that of the additional water molecule in the Fe-bound ApeSOD octahedral structure Thus, it can be assumed that the coordinated water in Fe-bound ApeSOD inhibits superoxide binding Although the polypeptide conformations of the Mn- and Fe-bound ApeSODs were almost the same (Fig 1A), a slight difference was observed in the side chain of Tyr39 (Fig 3D) This Tyr residue is conserved in Mn- and Fe-SODs, constitutes the outer sphere of the active site and has been shown by mutational studies to play a critical role in catalysis [29,30] The OH of Tyr39 was found to have shifted toward the apical water molecule upon Fe binding (Fig 3D) This shift was analogous to that of Tyr34 in E coli Mn-SOD upon binding of hydrogen peroxide to the central metal [26] Peroxide-bound E coli Mn-SOD represents a product-inhibited form of the reduction step from superoxide to hydrogen peroxide [26] These findings lead to the hypothesis that Fe-bound ApeSOD mimics the product-inhibited form and the shift of Tyr39 suppresses the release of the peroxide product This may be one of the reasons why ApeSOD is less active in its Fe-bound form It is noteworthy that this shift of the Tyr residue is not observed in the cambialistic SOD from P shermanii (Fig 3E), which exhibits almost the same activity in the presence of Mn and Fe as cofactors [24] Thermophilic bacteria, as well as thermophilic archaea, produce thermophilic enzymes Comparison of two thermophilic SODs, ApeSOD (an archaeon) and TthSOD (a bacterium) revealed that ApeSOD contained more ion pairs, especially intersubunit ion pairs, than TthSOD (Table 4) Because electrostatic FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 605 Crystal structure of SOD from A pernix K1 T Nakamura et al interactions are dominant under organic conditions, we expected that the difference in number of ion pairs would be reflected in a corresponding difference in the stability of the proteins in organic solvents Indeed, although the stabilities of the two SODs were indistinguishable in aqueous solution, ApeSOD was more stable than TthSOD in a solution containing ethylene glycol or ethanol (Fig 4) It is reasonable to conclude that the dominant electrostatic interaction in ApeSOD contributed not only to heat tolerance, but also to tolerance of the enzyme to organic environment For enzymes to be used in industrial applications, it is preferable that they have both structural and functional stability when placed under severe conditions Thus, because of its thermal stability and tolerance to an organic medium, ApeSOD shows promise as a potentially applicable enzyme Experimental procedures Protein expression and purification PCR was used to amplify the ORF of ApeSOD (APE_0741) from genomic DNA of A pernix K1 (NBRC 100138, provided by National Institute of Technology and Evaluation, Chiba, Japan) The PCR product was cloned into an expression plasmid vector pET11 (Novagen, Darmstadt, Germany) E coli Rosetta (DE3) cells harboring the expression plasmid were cultivated in LB medium containing 0.1 mgỈmL)1 ampicillin at 37 °C and protein expression was induced by the addition of mm isopropyl thio-b-d-galactoside (final concentration) The E coli cells were disrupted by sonication and the soluble proteins were subjected to streptomycin and heat treatments, as described previously [31] The resulting solution was dialyzed in 20 mm sodium acetate buffer (pH 4.8) and applied onto a cation exchange column (HiTrap SP; GE Healthcare, Piscataway, NJ, USA) The protein was eluted by a linear gradient of 0–1 m NaCl in the same buffer The fractions containing ApeSOD were collected, concentrated and gelfiltered with a Superdex 75 column equilibrated with 20 mm Tris-HCl (pH 8.1), with 150 mm NaCl as the final step The purified protein was dissolved in 20 mm Tris-HCl (pH 8.1) The protein concentration was determined from its absorbance at 280 nm [32] Gel filtration Analytical gel filtration chromatography was performed using a Superdex75GL (10 ⁄ 30) column (GE Healthcare) with a buffer containing 20 mm Tris-HCl (pH 8.1) and 150 mm NaCl The flow rate was 0.8 mLỈmin)1 The column was calibrated using a Gel Filtration Calibration Kit LMW (GE Healthcare) 606 Ultracentrifugation ApeSOD solution in 20 mm Tris-HCl (pH 8.1) containing 150 mm NaCl was subjected to a sedimentation equilibrium analysis using a Beckman Optima XL-A analytical ultracentrifuge with an An-60 Ti rotor (Beckman Coulter, Fullerton, CA, USA) Samples were centrifuged at 9500 g for 41 h at 20 °C The molecular mass of the protein was calculated from the sedimentation equilibrium plot Incorporation of metals Metal cofactors were incorporated into the enzyme by one of two procedures To incorporate metals during growth of bacterial host cells, mm MnSO4 (for Mn-bound ApeSOD) or mm FeSO4 (for Fe-bound ApeSOD) was added to the medium [33] The metal-containing enzymes were purified using EDTA-free buffers Alternatively, the metals could be incorporated into the purified enzyme For this technique, approximately mm ApeSOD in 20 mm TrisHCl (pH 8.1) was incubated with 10 mm MnSO4 or 10 mm FeSO4 at 70 °C for h The excess metals were removed by dialysis and the enzyme was further purified by gel filtration with a Superdex75 column equilibrated with 20 mm Tris-HCl (pH 8.1) and 150 mm NaCl The metal content of the enzyme was measured by inductively coupled plasma atomic emission spectrometry using a Perkin-Elmer Optima3300DV spectrometer (Perkin-Elmer, Waltham, MA, USA) Activity assay The activity of SOD was assayed with a SOD Assay KitWST (Dojin, Kumamoto, Japan) at 37 °C via the xanthine oxidase-WST-1 method [34] In accordance with the manufacturer’s instructions, one unit was defined as the amount of activity that caused 50% inhibition of WST-1 reduction Crystallization, data collection and processing ApeSOD was crystallized by hanging drop vapor diffusion, with a reservoir solution containing 100 mm HepesNaOH (pH 7.5) 10% (w ⁄ v) poly(ethylene glycol) 6000 and 8% (v ⁄ v) ethylene glycol at 20 °C The crystal was cryoprotected with modified reservoir solution containing 22.5% ethylene glycol, cooled in a nitrogen gas stream (100 K) and subjected to X-ray diffraction measurements with synchrotron radiation at SPring-8 (Harima, Japan) The collected data were integrated and scaled with hkl2000 [35] The initial phase was determined by molecular replacement with molrep in the ccp4 suite [36,37] Fe-SOD from S solfataricus [18] (Protein Data Bank code: 1WB8) was used as the search model The resulting structure was subjected to simulated annealing using cns FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS T Nakamura et al [38], followed by further refinement with refmac in the ccp4 suite [37,39] The secondary structure of proteins was assigned by dssp [40] Stereochemical analysis was performed using procheck [41] Ion pairs were analyzed using contact in the ccp4 suite [37] and confirmed visually using coot [42] Ion˚ pair interactions were identified using distances < A When we counted the interactions, we excluded all residues involved in the binding of the metal cofactor ApeSODs in different forms were superimposed with the least square fit of Ca atoms of the residues in the range 10–200 ApeSOD and P shermanii SOD were superimposed with secondary-structure matching [43] Thermal stability Heat treatment of ApeSOD (Mn-bound form) and TthSOD were performed in a buffer containing 50 mm Hepes-KOH (pH 7.0) and 0.5 mm MnSO4, with a protein concentration of 0.8 lm When indicated, the buffer contained 40% ethylene glycol or various concentrations of ethanol Thermal stability was evaluated by measuring enzyme activity after incubation Acknowledgements Diffraction data were collected at the Osaka University beamline BL44XU at SPring-8, equipped with MX225-HE (Rayonix), which is financially supported by Academia Sinica and National Synchrotron Radiation Research Center (Taiwan, ROC) We thank Ms M.Sakai (Osaka University) for performing ultracentrifugation analysis and Mr K Mieda and M Sakata (Tottori University) for technical assistance in enzyme assay This study was supported by a Grant-in-Aid for Scientific Research (21510237) from Japan Society for the Promotion of Sciences (JSPS) References Miller AF (2004) Superoxide dismutases: active sites that save, but a protein that kills Curr Opin Chem Biol 8, 162–168 Smith MW & Doolittle RF (1992) A comparison of evolutionary rates of the two major kinds of superoxide dismutase J Mol Evol 34, 175–184 Edward RA, Whittaker MM, Whittaker JW, Jameson GB & Baker EN (1998) Distinct metal 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geometrical features Biopolymers 22, 2577–2637 41 Laskowski RA, MacArthur MW, Moss DS & Thornton JM (1993) PROCHECK – a program to check the stereochemical quality of protein structures J Appl Crystallogr 26, 283–291 Crystal structure of SOD from A pernix K1 42 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics Acta Crystallogr D: Biol Crystallogr 60, 2126–2132 43 Krissinel E & Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr D: Biol Crystallogr 60, 2256–2268 44 DeLano WL (2002) The PyMol Molecular Graphics System DeLano Scientific, San Carlos, CA, USA FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 609 ... illustrates the superimposition of ApeSOD with the cambialistic SOD from P shermanii The overall structures of these enzymes were similar; the rmsd of the 747 Ca atoms was ˚ 0.695 A ApeSOD eluted from the. .. superoxide dismutases Biochim Biophys Acta 1804, 24 5–2 62 Crystal structure of SOD from A pernix K1 Sugio S, Hiraoka BY & Yamakura F (2000) Crystal structure of cambialistic superoxide dismutase from. .. inhibition of the binding of the superoxide substrate and the other to product inhibition by hydrogen peroxide There are two mechanisms proposed for the reaction cycle of Mn ⁄ Fe-SOD: the 5-6-5 mechanism

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