Tài liệu Báo cáo khoa học: The solution structure of reduced dimeric copper zinc superoxide dismutase doc

The solution structure of reduced dimeric copper zinc superoxide dismutase The structural effects of dimerization Lucia Banci, Ivano Bertini, Fiorenza Cramaro, Rebecca Del Conte and Maria Silvia Viezzoli Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, Italy The solution structure of homodimeric Cu 2 Zn 2 superoxide dismutase (SOD) of 306 aminoacids was d etermined on a 13 C, 15 N a nd 70% 2 H l abeled sample. Two-thousand e ight- hundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a f amily of 30 conformers with an rmsd from the average of 0.78 ± 0.11 and 1.15 ± 0.09 A ˚ for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop t o 0.65 ± 0.09 and 1.08 ± 0.11 A ˚ for the backbone and heavy atoms, respectively. The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural dif- ferences between the two subunits as well as between dif- ferent molecules in the unit cell. The elements of secondary structure, i.e. eight b sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the c ontrary, l oops IV and V I show some disorder. The residues of the active cavity are well defined whereas within th e various subunits of the X-ray structure some are disordered or display different orientation in dif- ferent X-ray structure determinations. T he copper(I) ion and its ligands are well defined. This structure t hus represents a well defined model in solution relevant for structure–func- tion analysis of the protein. T he comparison between the solution structure of monomeric mutants and the present structure shows that the subunit–subunit interactions in- crease the order in loop II. This has the consequences of inducing the structural and d ynamic properties that a re optimal for the enzymatic function of the wild-type enzyme. The regions 37–43 and 89–95, constituting loops III and V and the initial p art of t he b barrel and s howing several mutations in familial amyotrophis lateral sclerosis (FALS)- related proteins have a quite extensive network of H-bonds that may account for t heir low mobility. Finally, t he con- formation of the key Arg143 residue is compared to that in the other dimeric and monomeric structures as well as in the recently reported structure of the CCS–superoxide dismu- tase (SOD) complex. Keywords: superoxide dismutase; solution structure; dimeric protein; NMR; FALS. Cu 2 Zn 2 SOD is a well known homodimeric enzyme of 32 000 Da that catalyzes the dismutation of the superoxide radical to h ydrogen p eroxide a nd oxygen through a two s tep reaction [1–5]: Cu 2þ þ O À 2 ! Cu þ þ O 2 Cu þ þ O À 2 ! Cu 2þ O 2À 2 ÀÁ ! 2H þ Cu 2þ þ H 2 O 2 The active site of each subunit contains both a zinc and a copper ion, the latter being the site of the reaction. Copper occurs in the oxidized and in th e r educed state, both of which are necessary for the function. The X-ray structure of the oxidized form has been available since 1982 for the bovine enzyme [6,7] and several other structures have become available [8–18]. Reduced state structures are also available although the picture is less clear-cut around the copper-binding site [19–21]. Certainties on the protona- tion of His63, which b ridges Cu an d Zn in th e oxidized form but is protonated in the reduced form, come from 1 HNMR studies [22–25]. Eventually, monomeric forms were obtained through site-specific mutagenesis and t he NMR solution structure [26,27] as well as the crystal structure [28] of the reduced form were re ported. Also the b ackbone mobility of the monomeric state was investigated and compared with that of the dimeric species and it was concluded t hat, as far as motions in the ps to ns timescale are c oncerned, t he region c onsisting of residues 131–142, which forms one side of the a ctive site channel, is less mobile in the monomeric mutant than in the dimeric wild-type protein; structu ral fluctuations in this region have been suggested to play a role in assisting the superoxide anion in sliding towards the active site [29,30]. Moreover, the regions consisting of residues 47–59, 76–86 and 151–153, which are Correspondence to I. Bertini, Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy. Fax: + 39 055 4574271, Tel.: + 39 055 4574272, E-mail: bertini@cerm.unifi.it Abbreviations: SOD, superoxide dismutase; Q133M2SOD, F50E/ G51E/E133Q monomeric mutant superoxide dismutase; FALS, familial amyotrophis lateral sclerosis; M4SOD, F50E/G51E/V148K/ I151K monomeric mutant superoxide dismutase; CCS, yeast copper chaperone for superoxide dismutase; TPPI, time proportional phase increments. Note: The PDB ID code for the solution structure of homodimeric Cu 2 Zn 2 superoxid e dismutase is 1L3N. (Received 5 October 2001, revised 4 February 2002, accepted 16 February 2002) Eur. J. Biochem. 269, 1905–1915 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02840.x located at the subunit–subunit interface, were found to be more rigid in the dimer. This behavior was rationalized by considering that the presence of the second subunit produces residue–residue interactions, thus reducing their motions [30]. Also on the ms to ls time range, the subunit– subunit interface displays increased m obility in the mono- meric state with respect to the dimeric one. In particular, conformational equilibria were observed for residues around Cys57 a nd Cys146. T he former residue forms an H-bond with the guanidinium group of Arg143 [31], which is located in t he active site channel pointing towards the copper ion and whose side-chain orientation is optimized for correctly orienting the incoming superoxide an ion for the electron transfer process. The equilibrium between multiple conformations for this group and a different average structural orientation does not allow Arg143 to assume the optimal orientation for the enzymatic reaction [26,27,30]. T his c ould a ccount for the reduced enzymatic rates of the artificial monomeric species. The X-ray structures of the dimeric wild-type form of this protein show different structural details in the active site, both between the two subunits of the same molecule and among crystallographically independent subunits. This holds also for a number of loops [32]. The question of why SOD is a dimer and whether there is a cooperativity or anticooperativity between the two subunits in the physio- logical picture has never been completely solved. In this context we decided to solve the solution structure of the reduced dimeric protein (by using a classical NMR approach) in order to compare the solution structures of the monomeric and dimeric species as well as the solution and the crystal structures. The aim is to classify the effects of dimerization on the structural details. MATERIALS AND METHODS Sample preparation Dimeric human SOD was expressed i n Escherichia coli TOPP1 strain (Stratagene). The 15 Nand 15 N, 13 C, 2 H labeled proteins were obtained b y growing the cells in minimal m edium (M9) a s previously reported [30]. T he samples were isolated and purified according to previously published p rotocols [33]. The triple labeled dimeric SOD contained about 70% 2 H. Reduction of the copper ion was achieved by addition of sodium isoascorbate to a final concentration of about 4–6 m M ,in20 m M phosphate buffer at pH 5.0 u nder anaerobic conditions. The NMR samples had a concentration of about 2 m M in dimeric protein and contained 10% D 2 O for the lock signal. NMR experiments The NMR experiments were recorded on Bruker Avance 800, 700 and 600 spectrometers operating at 18.7, 16.4 and 14.1 T, re spectively . The assignment of the back bone is already available [30]. For the assignm ent of side chains H(C)CH-TOCSY, a t 600MHz,and(H)CCH-TOCSY,at800MHz[34],were performed, using 1024 ( 1 H) · 112 ( 13 C) · 256 ( 1 H) data points and spectral windows of 9258 Hz ( 1 H) · 11 184 Hz ( 13 C) · 9258 Hz ( 1 H) and 1024 ( 1 H) · 128 ( 13 C) · 280 ( 13 C) data points with spectral windows of 12 019 Hz ( 1 H) · 16 667 Hz ( 13 C) · 16 667 Hz ( 13 C), respectively. A 15 N-NOESY-HSQC and a 13 C-NOESY-HSQC [35] were collected at 800 MHz to obtain dipolar connectivities; a HNHA [36], at 700 MHz, and HNHB [37], at 800 MHz, experiments, were performed to determine the 3 J HNHa coupling constants and additional constraints for the v 1 torsion angles as well as stereospecific assignments for the Hb protons. The 15 N-NOESY-HSQC was recorded with spectral windows of 9569 Hz ( 1 H) · 2989 Hz ( 15 N) · 9569 Hz ( 1 H) for 2048 ( 1 H) · 88 ( 15 N) · 29 6 ( 1 H) data points. The 13 C-NOESY-HSQC was acquired with 1024 ( 1 H) · 112 ( 13 C) · 256 ( 1 H) data points with s pectral windows of 9615 Hz ( 1 H) · 19 230 Hz ( 13 C) · 9615 Hz ( 1 H). For both experiments the m ixing time was 130 ms. The three-dimensional HNHA experiment was carried o ut using s pectral windows of 9124 Hz ( 1 H) · 9124 Hz ( 1 H) · 3125 Hz ( 15 N) for 1024 ( 1 H) · 128 ( 1 H) · 32 ( 15 N) data points, the three-dimensional HNHB and the two- dimensional reference experiments were c arried out u sing spectral windows of 11 160 Hz ( 1 H) · 3244 Hz ( 15 N) · 11 160 Hz ( 1 H) for 1024 ( 1 H) · 48 ( 15 N) · 12 8 ( 1 H) and 11 160 Hz ( 1 H) · 3244 Hz ( 15 N) for 1024 ( 1 H) · 256 ( 15 N) data points, respectively. These experiments were collected at 296 K and they were performed using pulsed field gradients along the z-axis. Watergate two-dimensional N OESY experiments [ 38] at 296 K and at 286 K were registered at 800 MHz to identify connectivities involving h istidines of the active site. In both experiments 2048 ( 1 H) · 1024 ( 1 H) data points were acquired with spectral w indows 9124 Hz ( 1 H) · 9124 Hz ( 1 H); mixing times of 130 and 60 ms for t he experiments acquired at 296 and 286 K, respectively, were used. In order to detect the amide hydrogen–deuterium exchange a series of 1 H- 15 N HSQC spectra on the sample prepared dissolving the lyophilized protein in D 2 O solution were collected at 800 MHz, at 296 K. 1024 ( 1 H) · 256 ( 15 N) data points were acquired with spectral windows 12 019 Hz ( 1 H) · 4065 Hz ( 15 N) for each spectrum. Each 1 H- 15 N HSQC spectrum was acquired in 20 min every hour over a 24-h period. After 4 days from the dissolution in D 2 O one experiment was a cquired to d etect the r emaining amide protons. Quadrature detection in the indirect dimensions was performed and water suppression was achieved through the WATERGATE sequence [39]. Data were processed with the standard Bruker software packages ( XWINNMR ). Data analysis and assignment was performed using the program XEASY (ETH, Zurich, Swit- zerland) [40]. Structure calculation NOE cross-peaks in three-dimensional 15 Nand 13 C- NOESY-HSQC spectra and in two-dimensional N OESY spectra were integrated and c onverted into upper distance limits f or inte rproton distances with the program CALIBA [41]. The calibration curves for this conversion were adjusted iteratively as t he structure c alculations proceeded. N OE cross peaks due to couplings between the two subunits were converted into upper distance limits using specific calibra- tion curves. In the case of protons belonging to the ligand histidines an independent calibration has been used for each histidine. Stereospecific assignments of diastereotopic 1906 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002 protons have been obtained using the program GLOMSA [41] and by the analysis of the HNHB experiment. Backbone dihedral angle restraints / were derived f rom 3 J HNHa coupling constants by means of the appropriate Karplus relationship. For 3 J HNHa values larger than 7 Hz the / angle ranges between )155° and )80° while for values lower than 4.5 Hz it ranges between )70° and )30° [36]. Backbone dihedral angle w for r esidue (i ) 1) was deter- mined from the ratio of the intensity of the d aN (i ) 1,i)and d Na (i,i) NOE found on the 15 N plane of residue (i)inthe 15 N-NOESY-HSQC. Ratio values of the residue (i ) 1) larger than 1 are charact eristic of b sheets, with w values ranging between 60° and 175°, while values smaller than 1 indicate a right handed a helix, with w values between )90° and )10° [42]. v 1 torsion angle constraints were derived by the intensity ratios between the volume integral, d Nb (i,i), in the three-dimensional HNHB and the volume integral, d NH (i,i), in the two-dimensional reference spectra, as previously reported [37]. Structure calculations were performed using the program DYANA [43]. Fourteen-hundred random conformers were annealed using the above constrains in 18 000 steps for the initial calculations on a single subunit and in 22 000 steps for the dimeric form. The dimeric nature of the protein was taken into account by co nnecting the amino a cid sequence of the two subunits with a chain of linkers composed of atoms with a null Van d er Waals r adius. The metal ions were included i n the calculations by adding special linkers (pseudoresidues) in the amino-acid sequence following the same procedure already used for the monomeric forms [26]. The linkers only define the metal–nitrogen distances, leaving the conformation of the histidines completely free; the bond angles at the copper and zinc ions are not imposed but can freely change in the structural calculations, being only determined by the e xperimental intrahistidine NOEs. The presence of the disulfide bridge between Cys57 and Cys146 was checked through SDS/PAGE and through the analysis of the 13 C shifts of the Cb of the cysteines. In the calculations thr ee upper a nd three l ower distance limits were used to enforce t he disulfide bond Cys 57–Cys146 [between the Sc moieties of the two Cys, 2.0 (lower) and 2.1 (upper) A ˚ , and between the Cb of one Cys with the S c of the other, 3.0 (lower) and 3.1 (upper) A ˚ ][44]. The program CORMA [45], which is based on relaxation matrix calculations, was used to back calculate the NOESY cross-peaks from the calculated structure to assess the quality of the structure. The final family was made up of 30 structures with the lowest target function. Restrained energy minimization in vacuum (REM calculations) was applied to each member of the family using the program AMBER 5.0 [46]. The s etup of the program and the parameters for the metal ions are as previously reported [21]. T he value of NOE and torsion angle potentials h ave been applied with force constants of 50 kcalÆmol )1 ÆA ˚ )2 (NOE), 32 kca lÆmol )1 Ærad )2 (/, Y)and 2kcalÆmol )1 Ærad )2 (v 1 ). The program MOLMOL [47] was used for identification of hydrogen bonds (within the distance of 2.6 A ˚ between donor and acceptor, t he N-H-O angle larger than 140° and occurrence in at least 50% of the conformers). The quality of the structure has been estimated by Ramachandran plots obtained using the program PROCHECK - NMR [48]. RESULT AND DISCUSSION Resonance assignment Native dimeric SOD is co mposed of two identical subunits which produce degenerate resonances. Although this was already shown for the active site resonances from the investigation on the Cu 2 Co 2 SOD derivative [ 49], it i s a relevant result as X-ray data [32] often i ndicate different conformations for the two subunits [10]. T hus, averaging occurs on the NMR time scale, i.e. faster than seconds. The proton a nd 15 N resonances are not well dispersed and experience extensive overlap due to the specific folding of the protein, characterized by extensive b sheet structure. However, most of the 1 H- 15 N cross peak degeneracies present in 15 N-HSQC spectrum were resolved in at least one of the HNCA, HN(CO)CA, HNCO a nd HN(CA)CO- TROSY-type spectra already performed for the backbone assignment, reported by us [30]. The assignment of the resonances of the s ide chains was performed through the analysis of three-dimensional H(C)CH-TOCSY and (H)CCH-TOCSY spectra together with 15 N-NOESY-HSQC and 13 C-NOESY-HSQC spectra. In this way about 92% of the total proton resonances were assigned. All the backbone proton and nitrogen resonances, with the single exception of Phe64, were assigned. All the nitrogen side chain resonances o f Asn a nd Gln, with the exception of Gln153 (th e l ast o ne in each s ubunit), were assigned. Ninety-nine percent of the backbone 13 Creso- nances were assigned and about 86% of the 13 C side-chain resonances. All the ring protons of the histidines of the active site and o f His43 were ass igned through t he two- dimensional NOESY map. The histidine coordination mode was determined through 1 H- 15 N heteronuclear experiments, by detecting the 2 J 15 N- 1 H coupling between the imidazole nitrogen and nonexchangeable imidazole protons. Structure constraints and calculations In three-dimensional 15 N- and 13 C-NOESY-HSQC spectra and i n two-dimensional 15 N-NOESY s pectra, 356 6 NOE cross peaks were assigned and converted into distance constraints. Forty-nine dihedral / angles constraints were obtained from the analysis of th e HNHA spectrum, 52 dihedral w angles were obtained from the 15 N- NOESY-HSQC spectrum and 14 v 1 torsion angles from the HNHB spectrum. A total of 45 proton pairs were stereospecifically assigned with the program GLOMSA :16 protons belonging to bCH 2 ,11toaCH, 1 to cCH 2 ,1to dCH 3 and 1 to cCH 3 ;and15bCH 2 were assigned by the analysis of the H NHB experiment. In each subunit the metal ions were included by allowing copper t o bind to Ne2 of His48 and His120 and to Nd1of His46, and zinc to Nd1 of His63, His71, and His80 and to Od1 of Asp83. Lower and upper distance limits of 1.8 and 2.3 A ˚ , respectively, were imposed between t he metal i ons and the donor atoms. Finally, 3276 upper distance limits were generated of which 2853 were due to meaningful NOEs. All the available information on the system, the linewidth and the number of signals, lead to a dimeric species with twofold s ymmetry. The NOEs a nd 115 dihedral angles were initially used for the structural calculations of the Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1907 monomeric species. During these initial s tructure calcula- tions, the presence of a small but significant number of NOEs (96) inconsistent with couplings with protons o f residues of the same subunit, were identified and assigned to connectivities between protons belonging to two differ- ent subunits. They w ere introduced in the calculations at a later stage after r efinement of the monomeric structure. Only one NOE has a contribution from both inter and intra subunit; no severe violation with respect to the calibration was observed. For the calculations of the dimeric s tructure, the intra s ubunit NOEs and dihedral angle constrains were duplicated for each subunit and the inter subunit NOEs were included. The number of constraints, divided in classes, are listed in Table 1. The number of experimental N OEs per residue per subunit is reported i n Fig. 1. From the final calculations, a family of 30 conformers, with the l owest target f unction of 5.02 A ˚ 2 (average value 5.99 A ˚ 2 ), was obtained with a n average violation p er residue of 0.016 A ˚ . E ach c onformer o f t his f amily was refined further through REM calculations. The rmsd to the mean structure of the family is 0.79 ± 0.11 and 1.35 ± 0.09 A ˚ for the backbone and the heavy atoms, respectively (rmsd calculated over the fragment 3–151 for the holo protein). After the refinement, the rmsd values a re 0.78 ± 0.11 and 1.15 ± 0.09 A ˚ for t he backbo ne and the heavy atoms, respectively. If the rmsd is evaluated for each subunit of the protein the values drop to 0.65 ± 0.09 and 0.66 ± 0.10 A ˚ , respectively, for the backbone of the two subunits. The difference between rmsd of monomeric and dimeric species is due to the indetermination of the reciprocal orientation of the two subunits. The average total penalty for the REM family of the dimeric p rotein is of 1.42 ± 0.07 A ˚ 2 for the distance constrains; while for the average structure the value is 1.36 A ˚ 2 . The rmsd values per residue, with the respect to the average structure, are shown in Fig. 2. General shape of the protein and comparison with X-ray structures A tube representation of the family of structures (back- bone and metal ions only) is shown in Fig. 3. The family of conformers was analyzed with PROCHECK - NMR and the results of t he analysis are reported in Table 1. The secondary structure elements are eight antiparallel b strands and a short five-residue a helix, which, connected by loop regions, produce the typical S OD Greek key fold. The secondary structure part of the protein is well defined. The average rmsd values for the segments involved in the b barrel are 0.50 ± 0.08 A ˚ and 0.85 ± 0.06 A ˚ for the backbone and all heavy atoms, respectively, which indi- cates that the b strands are characterized by lower disorder than the loops connecting them. If a s ingle subunit is considered, the bbarrel rmsd values are 0.38 ± 0.06 A ˚ and 0 .7 7 ± 0.06 A ˚ , f or the backbone and a ll heavy atoms, respectively. These values indicate that the b strands in each subunit are well defined. The a helix within the family of confor mers has an average rms d to Table 1. Restraint violations and structural and energetic statistics for the solution structure of reduced human SOD. RSM violations per experimental distance constraint (A ˚ ) b REM a (30 structures) <REM> a (mean) Intraresidue (723) 0.0251 ± 0.0013 0.0245 Sequential (1546) 0.0124 ± 0.0010 0.0119 Medium range (924) c 0.0149 ± 0.0009 0.0140 Long range (2513) 0.0115 ± 0.0005 0.0109 Total (5706) 0.0147 ± 0.0004 0.0147 RSM violations per experimental dihedral angle constraints (deg) b Phi (98) 1.55 ± 0.21 1.54 Psi (104) 0.52 ± 0.24 0.0 Chi1 (14) 0.41 ± 0.26 0.0 Average number of violations per structure lower than 0.3 A ˚ Intraresidue 51.4 ± 3.8 51 Sequential 42.7 ± 4.5 41 Medium range 31.9 ± 3.3 26 Long range 52.8 ± 3.7 53 Total 178.7 ± 6.3 171 Phi 8.1 ± 1.9 8 Psi 1.5 ± 0.9 0 Chi1 1.3 ± 0.6 0 Average no. of NOE violations larger than 0.3 A ˚ 00 Structural analysis d % of residues in most favourable regions 71.6 73.6 % of residues in allowed regions 25.6 24.4 % of residues in generously allowed regions 2.3 2.1 % of residues in disallowed regions 0.6 0 a REM indicates the energy minimized family of 30 structures, <REM> is the energy minimized mean structure obtained from the coordinates of the individual REM structures. b The number of experimental constraints for each class is reported in parentheses. c Medium range distance constraints are those between residues (i,i +2)(i,i +3)(i,i + 4) and (i,i + 5). d As it results from the Ramachandran plot analysis. 1908 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the mean structure of 0.39 ± 0.01 A ˚ and 0.91 ± 0.21 A ˚ , for the backbone and all heavy atoms, respectively. These values drop to 0.16 ± 0.08 A ˚ and 0.65 ± 0. 26 A ˚ when a single subunit is c onsidered. The c omparison of the present structure with the X -ray structures of the human oxidized protein ( 1SOS) [11] and its G 37R mutant [50] show that the protein has the same folding in solution and in solid state. The loops connecting the secondary s tructure elements can be divided in two groups: the loops I, III and V are quite well defined, while loops II, IV and VI are more disordered. The odd loops are located on the opposite side of the barrel with respect to region involved in the subunit–subunit interface. The even loops are in part located at the subunit– subunit interaction. The first part of loop IV (49–62) shows (Fig. 4 ) a much lower backbone rmsd in the present Fig. 1. Number of intraresidue (white), sequential (light grey), medium-range (grey) and long-range (black) intra subunit NOEs per residue (bottom) and number of inter subunit NOEs per residue (top) in human reduced native SOD. Fig. 2. Average rmsd values of backbone (j) and heavy atom (h) on two subunits per residue with respect to the average structure of human reduced native SOD (bottom). Backbone (j) and heavy atom (h) rmsd values of a single subunit per residue with respect to the average structure of human reduced native SOD (top). Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1909 structure than in t he monomeric Q133M2SOD structure. This can be related to the occurrence of interactions with the other s ubunit that m inimize the exposure to solvent of residues at the interface and stabilizes a single conformation for them. Indeed, in the segment 50–59 of Q133M2SOD, five backbone HN signals were not assigned, probably due to line-broadening as a consequence of p roton exchange with solvent due to their surface location. This is consistent with the analysis o f t he amide h ydrogen–deuterium e x- change behaviour previously reported [51]. The other loops are still disordered even in the dimeric form. Therefore, this behaviour suggests that this is a feature typica l of this part o f the protein independent of its quaternary structure. The change in conformation of loop 50–59 upon dimerization is reflected also on the location of Cys57, which can or cannot perform a H-bond with the side chain of Arg143 depending on its conformation (see below). FALS mutations are spread over the entire molecule but a higher density of mutations are clustered in a few regions of the protein: at the interface between the two subunits (mainly in loop IV and b 8), in the odd loops and at the corresponding end of the b barrel, and in the even loops [52–56]. Some of the residues involved in FALS m utations are conserved i n SOD structures from different species (Fig. 5) [11]. The FALS mutations located in the first region are thought [11] to significantly destabiliz e the subunit– subunit contacts. This is in agreement with the NMR d ata Fig. 5. Close-up of one subunit of human reduced SOD showing FALS mutations. The F ALS mutations located in odd loo ps are shown in gray, those in b strands are in black and located in the region close to the subunit–subunit interaction are coloured black and the residue labels are underlined. Fig. 3. Tube representation of the family of 30 structures of human reduced native SOD obtained with DYANA calculations and refined with REM calculations. Elements of secondary structure are highlighted (gray, b structure; black, a struc ture). The drawing has been produced with MOLMOL [47]. Fig. 4. Comparison o f rmsd values to the average structure for the backbone between dimeric SOD (s) and E133QM2SOD (.). 1910 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002 on the solution structures [27,57] and on mobility studies [30] of monomeric variants and human dimeric SOD, where it has been shown that the absence of interactions with the other subunit has sizable effects on enzymatic stability and activity. Hydrogen–deuterium exchange A total of 104 amide protons out of 147 were still present in the 1 H- 15 N HSQC spectrum acquired 6 h after the disso- lution of the lyophilized sample in D 2 O. Fifty-one residues are located in regions having a defined secondary structure as they are involved in an extensive H-bond networks which stabilize the b barrel structure typical of this protein. Few exceptions are observed in one of the b sheets (b6) where amide protons belonging to three residues (Asp96, Ser98, Glu100) out of six, exchange within 40 min and those belonging to Asp101 within 1 h 40 min. Also the a helix shows exchanging amide protons in the time range between 40minto12h. After 4 days 85 peaks, mostly belonging to the b barrel and to loops III and VII were still present. Metal sites In Fig. 6 the active site is shown and compared with that of oxidized human SOD. All the metal ligands are well defined in a single conformation. For all the ligands, the rmsd value calculated for all heavy atoms is smaller than 0.8 A ˚ ,avalue that is similar t o t hat obtained for secondary structure elements. The ligand conformation is also very close to that observed in all the structures available for eukaryotic SOD, either based on X-ray or NMR analysis in solution, dimeric or monomeric. The only exceptions are His63 and t he copper ion. His63 experiences a l arger variability among the various structures and its orientation i s dependent on the copper oxidation state. I n oxidized SOD, His63 is coordi- nated to the Cu ion through its Ne2, the distance between copper and Ne2 being about 2.1 A ˚ in the human structures (1SOS) [11] and about 2.7 A ˚ in a mutant (G37R) [50]. In the reduced state, the bond between Cu and His63 is broken, producing an increase in distance between the two. In the case of the reduced dimeric yeast enzyme this distance increases to 3.2 A ˚ [20]. In the present structure the reduced copper is clearly tricoordinated, as expected from the data on the monomer. Indeed, upon copper reduction His63 becomes p rotonated at t he Ne2 position, the bound proton resonating at 12.3 p.p.m. and the distance between copper and Ne2being3.3A ˚ . In the present structure the major structural changes induced by copper reduction is the movement of the copper ion which moves away from His63, experiencing a displacement of about 1.7 A ˚ with respect to the oxidized enzyme. So, the increased Cu–His63 distance, in the reduced state, is due to a movement of copper more than to a change in conforma- tion of His63. The position of the other metal ligands, in the present structure, is very close to that found in the reduced dimeric yeast isoenzyme [20], whereas the copper ion positions in the two structures differ by about 1.0 A ˚ .It should be noted, however, that in the reduced yeast structure Cu a nd Ne2 of His63 are at a distance shorter than the sum of their van der Waals radii. The Zn ion does not experience significant movement from its site compared to the other structures. In reduced monomeric human mutants (Q133M2SOD and M4SOD) the Zn ion moves farther from the copper ion. The distance between the metals in the present structure is 7 A ˚ ,thisis similar to that in the dimeric yeast isoenzyme (6.7 A ˚ ), while in the human oxidized structure it ranges between 6.1 A ˚ to 6.3 A ˚ . About the active site channel The active site channel is located between the electrostatic loop VII (120–144), implicated in assisting and increasing the affinity for the active site of substrate, and loop IV (49– 82). A network o f H-bonds between the s ide chains o f some residues belonging to loop VII plays a crucial role in increasing the diffusion rates of the superoxide radical inside the cavity [33]. Comparing X-ray structures (1SOS, G37R and 1JCV) with the present one, it can be observed that the o rientation of the a helix is the s ame i n a ll the structures and the backbone remains almost unaltered. In contrast the side chains experience different conformations: Glu132 shows a different orientation in each of the structures and in each of the subunits in the crystal cells, whereas n o meaningful comparison can be carried out for Glu133, which shows disorder in the side chain. Ser134 and T hr135 are quite ordered in the present structure, but they have a different orientation of t he hydroxyl group with respect to the X-ray structures. Side chain of Lys136 has different orientations in each of the X-ray structures. The present one is closer to that in 1SOS and G 37R structures. Thr137 shows no significant changes in the orientation of th e side chain although a movement towards Arg143 is observed, which slightly decreases the width of theactivesitechannel. Thr58 and Glu133, with Glu132, define the opening of the active cavity. The width is about 13 A ˚ (distance between Thr58 Cc and Glu133 Oe), which is d ecreased by % 1A ˚ with respect to 1SOS and % 2A ˚ with respect to G37R. Arg143 with Thr137 form a ÔbottleneckÕ for the active site, which excludes sterically large nonphysiological anions. In Fig. 6. Active site of the family conformers of the reduced human dimer (blue) and of the oxidized human dimer (red). Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1911 the present structure also these residues are slightly closer than the X-ray struct ure. Arg143 i s important in orienting the superoxide anion towards the Cu ion. Comparing the present structure with 1SOS, G37R and 1JCW, the side chain of Arg143, in most of the conformers shows no significant changes in the orientation, while in the case of the monomers (Q133M2SOD and M4SOD) the Arg143 side chain has a different orientation (Fig. 7). Cys57, with residues 58 and 61, was proposed to stabilize the orientation of Arg143 [26,27], as a result of hydrogen bonds between the side chain of Arg143 (protons of Ng1andNg2 groups) and backbone carbonyls of Cys57, Thr58 and Gly61. The H-bonds involving C ys57, Thr 58 a nd Gly61 a re present in several conformers. The latter three residues are defined by 23, 24, 20 NOEs, respectively. Furthermore, Cys57 forms a disul- fide bond with Cys146, which is defined by 36 NOEs. Side chains of Arg143 and Cys57 are defined by 26 and seven NOEs, respectively. The Cu–Ng1andCu–Ng2 average distances of Arg143 are 7.2 and 7.3 A ˚ from copper, while in the oxidized human protein the distances are 5.8 and 7.0 A ˚ . This is consistent with the already discussed movement of the copper ion upon reduction. Cys57 seems to play a fundamental role in the process of copper transfer from the copper chaperone for SOD (CCS) and SOD itself as shown by t he recently solved structure of the CSS–SOD complex [58]. In the latter structure, Cys229 of CCS forms a disulfide bond with Cys57 o f SOD [58], which t herefore is not interacting any longer with Arg143. The guanidinium group of the latter residue in the complex is very far away from the site where copper should b e introduced and is pointing t owards the c haperone. The conformation of Arg143 is extremely sensitive to the position of Cys57 [26,27]. In the monomeric species, where Cys57 experiences conformational equilibria, still maintain- ing the disulfide bond, Arg143 is further from c opper than in the wild-type protein but closer than in the copper-free SOD in the complex. In the present solution structure of wild-type SOD where the Cys57 is quite rigid, Arg143 assumes the optimal conformation respect to the c opper. Therefore it seems that Arg143 is experiencing a movement that leads it to assume the correct conformation when SOD is passing from the complex with CCS (where SOD is in a monomeric state) to the single monomeric protein, to the final dimeric structure. Relevant H-bonds A network of H-bonds in dimeric human oxidized SOD [8] was proposed to play an important role in building the Greek key structure and in designing the metal binding site and the active cavity of the system. The analysis of the H-bonds in the present structure has been carried out with MOLMOL program [ 47] on the final structure. Except a few cases d iscussed later the hydrogens involved in H-bonds do not exchange in D 2 O. Some of the H-bonds present in the human oxidized X-ray structures are observed also in the solution structure. The H-bonds among ring hydrogens of His43 and backbone carbonyls of Thr39 and the Cu-ligand His120 ar e present in almost all the conformers of the family. Ho wever the He2 of His43 do exchange in D 2 O indicating solvent exposure of such H-bond. H-bonds involving ring hydrogens of His43 are important in linking the loop III to the b barrel and the active site. The presence of these H -bonds is co nsistent with th e N MR observation of two HN ring protons signals for His43 (which is not involved in metal binding) at pH 5.0. In the present structure, the side c hain Od of Asp124 forms a H -bond with the ring hydrogens He2 of His71 and of His46. Asp124 constitutes a long-range bridge between the copper site and the zinc site [8]. Mutations of residue 124, which have been found in FALS proteins, affect mainly the zinc site and its affinity for the zinc ion [59]. These mutations might produce zinc deficient species that h ave b een s hown to gain peroxynitrite producing a ctivity, a possible c ause of the FALS diseas e [ 60–63]. A conserved H -bond between backbone HN of His71 and CO of Thr135, important in stabilizing the active site channel, is present in several conformers. Thr135 belongs to the six residue helix involved in the recognition and in the electrostatic guidance of the superoxide anion. The amino acid site chains of Glu132, Glu133, Lys136 and Thr137 are involved in a hydrogen bonding network [33]. In the present structure this H-bond network is maintained. For the FALS mutations located in the region constituted by odd loops and one end of b barrel (Fig . 5), as for example G37R, the absence of some H-bonds in the b hairpin region (loop V) is supposed to be responsible of the misfunction of the enzyme [50]. In the present structure all the odd loops are well defined and this is consistent with the presence of a network of H-bonds that stabilizes this part of theprotein.ThisregioniscenteredonLeu38,calledthe ÔplugÕ of one end of the bbarrel [ 11], which fills a cavity formed by an array of apolar aminoacids present in different b strands (Ile35, the ring face of His43 and Leu144) and loop I (Val14). T hus producing a packed arrangement, crucial for correct enzymatic f unction and protein stability [64]. Conserved H-bonds observed in this crucial part of the protein, observed in the present structure and identified with the program MOLMOL , are summarized in Table 2. The H-bond connecting loop III and loop V, containing b hairpin (HN of Leu38 and CO of Gly93) and the H-bond between HN of Gly93 and CO of Asp90 (loop Fig. 7. View of the active channel of human reduced native SOD. Ori- entation of the s ide chain of Arg143 is reported for t he re duced human dimer (blue), for the oxidized h um an dimer (red), for t he Q133M2SOD (cyan) a nd for the reduced e nzyme derived from yeast (yellow). The Cu ion is shown as a sphere and the a helix is in orange. 1912 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002 V) and between HN of Asp92 a nd side chain carboxylic group of Asp90 are well conserved in all conformers of th e present family and in the X-ray structures (1SOS) even if some difference in the stability of H-bond could be present. Indeed the amide proton of Asp92 disappears in D 2 Oafter about 2 h, whereas the others a re still present four days after the dissolution in D 2 O. In the F ALS muta nt G37R t he H-bond between HN of Asp92 and the carboxylic group of Asp90 is p resent in only one of the two subunits [50]. The loss of this hydrogen bond in the G37R mutant [50] was proposed to allow a n increase fl exibility in t he b hairpin, with respect to the wild-type protein; the latter, in fact, is characterized by the absence of motions in the ps-ns timescale in this region [30]. Gly41, Gly37 and Gly93 seem necessary to support main chain conformations and the packing interaction in the h ydrophobic plug [64]. Gly41 is involved in H-bond with Ala89 that, in its t urn, is close t o the b-hairpin which i s further stabilized by the H -bond between Asp90 and Val94. The presence of extensive H-bond networks seem to play a fundamental role in stabilizing the secondary and tertiary structure of the protein. CONCLUSIONS The solution structure of dimeric human reduced Cu 2 Zn 2 SOD was determined to a satisfactory degree of resolution. The two monomers are identical on the NMR t ime scale. The elements of secondary structure are the same as in the X-ray structures and well resolved as well as the three odd loops and t he first part of loop IV, at t he inter–subunit interface. The even l oops, have a r elatively high r msd. A similar behavior i s observed in a recently reported X-ray structure of bovine SOD [32]. The structure i s also similar t o the solution structure of the monomeric mutants with the exception of the s ignificantly better definition of the first part (49–63) of loop IV, which is disordered in the monomers and experiences significant local mobility. T he active channel is formed by the electrostatic loop VII, where charged residues important in catalysis lie, and loop IV, where Cys57 is located. Upon dimerization, loop IV looses the conformational exchange equilibria, occurring in the ms-ls time range, and assumes a conformation which f avors the formation of the hydrogen bond network. The optimal conformation of the side chain of Arg143 is ensured by the formation of H-bonds between its terminal guanidinium group and the backbone oxygen atoms of Cys57 and Gly61 (loop IV). The latter network contributes to determine the optimal orientation o f the strategic residue Arg143 and reduces its mobility i n the subnanosecond time sc ale. In contrast, the increased mobility, in the subnanosecond time scale, of the electrostatic loop VII (a helix) could assist O 2 – in sliding inside t he active cavity, w here it reaches the correct position helped b y t he interaction with the correctly oriented Arg143. The optimal orientation of Arg143 is found also in a wild-type bacterial SOD [65], which i s naturally mono- meric and where Cys57 (human numeration) is still H-bonded to Arg143. The copper site in the present d imeric structure i s in a position similar to that of the monomeric mutants. Because X-rays, when irradiating the crystals, may change the oxidation state or the s olid state a nd may i nduce subtle structural changes, the present characterization of the reduced active site represents a f urther reliable p icture of the reduced protein. Upon reduction, copper moves inside the active cavity. This is consistent with the earlier proposal [20,28,66] that the superoxide ion hardly reaches copper (I) but rather interacts with the e2protonofHis63andis activated by this interaction for t he transfer of one electron from copper (I). Finally, the strong H-bond network involving odd loops and one end of the b barrel (Table 2) is observed in solution. It may be relevant that some FALS mutants disrupt this network, giving them the capability of catalyzing other toxic reactions. In conclusion, the present structure of the dimeric wild- type SOD, although at lower resolution with respect to the X-ray structures, provides a clear refined picture of the relevant residues i n solution a nd allows a thorough under- standing of the effects of establishing a quaternary structure. ACKNOWLEDGEMENTS This work was supported by the European Com munity ( Contract number HPRI-CT-1999-00009 and QLG2-CT-1999-01003), by Italian CNR (Progetto Finalizzato Biotecnologie 99.00286.PF49 and 99.00950.CT03) and by MIUR-ex 40%. REFERENCES 1. Fridovich, I. (1974) Superoxide dismutase. Adv. Enzymol. 41 , 35–97. 2. Fridovich, I. (1986) Superoxide dismutase. Adv. Enzymol. Relat. Areas Mol. Biol. 58, 61–97. 3. Valentine, J.S. & P antoliano, M.W. (1981) Protein–metal ion in- teractions in cuprozinc protein (superoxide dismutase). In Copper Proteins. 8 (Spiro, T.G., ed.), pp. 291–291. Wiley, New York. 4. Halliwell, B. & Gutteridge, J.M. 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S2 Experimental NOE intensities of reduced human SOD Table S3 v1 torsion angle constraints for a single subunit in reduced human SOD Table S4 / torsion angle constraints for a single subunit in reduced human SOD Table S5 w torsion angle constraints for a single subunit in reduced human SOD Table S6 Stereospecific assignments diastereotopic pairs for a single subunit in reduced human SOD . The solution structure of reduced dimeric copper zinc superoxide dismutase The structural effects of dimerization Lucia Banci,. to solve the solution structure of the reduced dimeric protein (by using a classical NMR approach) in order to compare the solution structures of the monomeric