Structural and dynamic aspects related tooligomerization of apo SOD1 and its mutantsLucia Bancia,b, Ivano Bertinia,1, Mirela Bocaa, Vito Calderonea, Francesca Cantinia, Stefania Girottoa, and Miguela Vierua

aMagnetic Resonance Center and Department of Chemistry, and bFiorGen Foundation, University of Florence, Via Luigi Sacconi 6,50019 Sesto Fiorentino, Italy

Edited by Angela M. Gronenborn, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved March 4, 2009 (received for reviewOctober 2, 2008)

The structural and dynamical properties of the metal-free form ofWT human superoxide dismutase 1 (SOD1) and its familial amyo-trophic lateral sclerosis (fALS)-related mutants, T54R and I113T,were characterized both in solution, through NMR, and in thecrystal, through X-ray diffraction. We found that all 3 X-raystructures show significant structural disorder in 2 loop regionsthat are, at variance, well defined in the fully-metalated structures.Interestingly, the apo state crystallizes only at low temperatures,whereas all 3 proteins in the metalated form crystallize at anytemperature, suggesting that crystallization selects one of themost stable conformations among the manifold adopted by theapo form in solution. Indeed, NMR experiments show thatthe protein in solution is highly disordered, sampling a large rangeof conformations. The large conformational variability of the apostate allows the free reduced cysteine Cys-6 to become highlysolvent accessible in solution, whereas it is essentially buried in themetalated state and the crystal structures. Such solvent accessibil-ity, together with that of Cys-111, accounts for the tendency tooligomerization of the metal-free state. The present results sug-gest that the investigation of the solution state coupled with thatof the crystal state can provide major insights into SOD1 pathwaytoward oligomerization in relation to fALS.

amyotrophic lateral sclerosis � NMR � X-ray � mobility � H2O/D2O exchange

More than 100 different variants of human copper-zincsuperoxide dismutase (Cu2Zn2 SOD) have been identified

and linked to the neurodegenerative disease familial amyotro-phic lateral sclerosis (fALS) by a gain-of-function mechanism (1,2). Although the mechanism of the toxicity is unknown, aberrantSOD1 protein oligomerization has been strongly implicated indisease causation (3, 4). Several recent publications (5, 6) havepresented compelling evidence that in vivo abnormal disulfidecross-linking of ALS mutant SOD1 plays a role in this oligomer-ization, and disulfide-linked SOD1 multimers have been de-tected mainly in mitochondria of neuronal tissues of SOD1-linked fALS patients and transgenic mice (7–9).

WT human SOD1 is an exceptionally stable, homodimeric32-kDa protein, located mainly in the cytoplasm, but it is alsopresent in the peroxisomes, the mitochondrial intermembranespace, and the nucleus of eukaryotic cells (10, 11). Each subunitof the dimer binds 1 copper and 1 zinc ion and folds as an8-stranded Greek-key �-barrel that is stabilized by an intrasu-bunit disulfide bond (Cys-57, Cys-146) near the active site (12).In vivo, in the highly reducing cytoplasm environment, theexistence of this intrasubunit disulfide bond points to its very lowreduction potential.

In addition to the 2 cysteines involved in the formation of theintramolecular disulfide bond, 2 reduced cysteines, Cys-6 andCys-111, are located on �-strand 1 and loop VI of WT humanSOD1, respectively. Among the loops connecting the 8 �-strands,2 have structural and functional roles. The electrostatic loop(loop VII, residues 121–144) contains charged residues thatcontribute to guiding the negatively-charged superoxide sub-

strate toward the catalytic copper site. The long zinc loop (loopIV, residues 49–84) contains all of the zinc binding residues.

We have recently reported (13, 14) that oxidized WT SOD1and several of its mutants, only when they are in the metal freeform (apo), give rise, in vitro, to soluble oligomers under aerobicconditions when the proteins are kept at 37 °C and at a concen-tration and pH close to physiological, i.e., 100 �M and pH 7. Theresulting soluble oligomers are formed by intermolecular disul-fide covalent bonds, involving Cys-6 and Cys-111, and by non-covalent interactions between �-strands, forming amyloid-likestructures capable of binding Thioflavin T (14). The rates ofprotein oligomerization are different for the various mutants,but eventually they give rise to the same type of solubleoligomeric species.

SOD1 enters as apoprotein the mitochondria, which are moreoxidizing cellular compartments (15, 16). The soluble oligomericspecies, formed through an oxidative process, might representthe precursor toxic species, whose existence would also suggesta common mechanism for ALS and fALS.

To investigate the mechanism for SOD1 oligomerization, thestructural and dynamic features of the metal-free state of SOD1 forboth WT and some ALS-related mutants were characterized bothin solution, through NMR, and in the crystal, through X-raydiffraction. We found that the metal-free state is significantlydisordered in the crystal for 2 pathogenic SOD1 mutants asreported for the WT protein (17, 18), at variance with that observedin the metalated state. In solution, the highly-disordered anddynamical metal-free state allows the free cysteines to becomeaccessible for oxidation and subsequent oligomerization, at vari-ance with what occurs in the metal-bound form.

Results and DiscussionIn the present study we characterized the apo form of WT SOD1and 2 ALS-related mutants with 2 extreme behaviors in terms ofoligomerization rates: T54R oligomerizes with rates slightlyslower than WT SOD1, whereas I113T has an oligomerizationrate more than twice that of WT SOD1 (14).

In the crystal structures of the metal-free form of WT SOD1and its mutants T54R and I113T, the asymmetric unit contains2 biologically relevant dimers (A–B and C–D). Although one hasa very well-defined electron density throughout the entire se-quence, the other dimer has clear breaks in the electron densityin the regions encompassing residues 68–78 (loop IV) andresidues 125–140 (loop VII). Overall, the structures are very

Author contributions: L.B., I.B., V.C., F.C., and S.G. designed research; M.B., V.C., F.C., S.G.,and M.V. performed research; L.B., I.B., M.B., V.C., F.C., S.G., and M.V. analyzed data; andL.B., I.B., V.C., F.C., and S.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 3ECU, 3ECV, and 3ECW).

1To whom correspondence should be addressed. E-mail: ivanobertini@cerm.unifi.it.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809845106/DCSupplemental.

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similar to each other. Only few minor local differences in termsof conformation, buried surface areas, and residues involved inH-bond interactions are detected (Table S1). Peculiar for apoT54R SOD1 is a mutation-related hydrogen bond, which isformed at the dimer interface, i.e., between NH2 of Arg-54 ofeach monomer and OD1 of Asn-19 of the other monomer in thesame dimer. This interaction might lead to a partial stabilizationof the dimeric state with respect to the WT protein and mightcorrelate with the slightly slower oligomerization rates for apoT54R compared with the WT protein (14), because oligomer-ization presumably occurs through monomerization (19). Thefree Cys-6, in all 3 apo SOD1 structures, is essentially buriedbecause of the constraints imposed by 2 H bonds with Ile-18,whereas the other free cysteine, Cys-111, has a considerably high(75%) solvent-exposed side chain (thiol group).

The 3 structures determined here clearly resemble the alreadyavailable partially apo (20% zinc in 1 of the 2 dimers) WT SOD1structure [Protein Data Bank (PDB) ID code 1HL4] (18).Similarly, the same electron density breaks were found in only 1of the 2 dimers. On the contrary, the fully metalated (Cu2,Zn2;holo) structures of both WT SOD1 [PDB ID code 1HL5 (18)]and I113T SOD1 mutant [PDB ID code 1UXL (20)] show awell-defined electron density throughout the entire sequence for

each dimer present in the asymmetric unit. The backbone rmsdbetween the holo and apo form of both WT and I113T SOD1 is�0.45 Å. The main structural differences between the holo andapo states are observed in the loops connecting the �-strands,where the electron density is broken in one of the dimers in thestructures of the apo state. Similar electron density breaks werealso observed for the structure of the apo state of the H46RSOD1 mutant (21), the only, up to now published structure of acompletely metal-free SOD1 mutant. On the contrary, thestructures of ALS-related SOD1 mutants, in the fully metalatedstate are very similar to each other, and particularly well orderedthroughout the sequence (20–25).

WT SOD1 and its mutants, T54R and I113T, in the apo state,form a continuous, extended arrangement of �-barrels stackedup along a direction (crystallographic b-axis) perpendicular tothe dimer interface (Fig. 1A). The tetramer A�B�C�D com-prising the asymmetric unit is surrounded by 4 others in the acplane. The intermolecular contacts between the 2 dimers in theasymmetric unit are between monomers A and D and monomersB and C. The H-bonding connections are different for the 2 pairs(Fig. 1B). A H-bonding network is also present between mole-cules in adjacent asymmetric units, with 7 strong and 3 weak Hbonds between monomers A and D1 (i.e., monomer of an

Fig. 1. Packing of molecules in apo WT SOD1. (A) The apo protein has an extended sheet of �-barrels arranged in the ac plane. Monomer A is shown in blue,monomer B is in yellow, monomer C is in light green, and monomer D is in violet. (B) Contacts between neighboring dimers: H bonds involving OD1 and ND1of Asn-26 and the O atom of Pro-66 are symmetrically present between monomers A and D. ND1 atom of Asn-26 of monomers C forms an H bond with the Oatoms of Val-103, Ile-104, and Ser-102 of monomer B. H bond is shown between OE2 atom of Glu-24 and OD2 atom of Asp-109. H-bonding network is presentbetween molecules in adjacent asymmetric units: the O atom of Lys-128 in monomer A forms H bonds with the N and O atoms of Asn-86 of monomer D1. TheO atom of Gly-129 has H bonds to the O atom of Asn-86 and to the OG1 atoms of Thr-88. The N and O atoms of Gly-130 participate in 3 H bonds: to Asn-86 Oand to Ser-98 N and O atoms, respectively. Finally, the N atom of Asn-131 forms a weak H-bond to the O atom of Ser-98. (C) The view obtained after rotatingA twice by 90° showing the zigzag arrangement of the constituent �-strands aligned along the long ac diagonal of the unit cell.

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adjacent asymmetric unit) and with 6 strong and 2 weak H bondsbetween monomers B and C1 (Fig. 1B). The view orthogonal tothe crystallographic b-axis shows that the apo WT SOD1 �-bar-rel forms a zigzag array of filaments (Fig. 1C). This behavior issimilar to that already observed for apo WT SOD1 reported byStrange et al. (18) and is common to amyloid-like fibrils (26).

The comparison between the structures of WT SOD1 andALS-related mutants was unable to shed light on the structuralbasis of different behaviors in oligomerization rates between WTand pathogenic mutants, although it did point at some confor-mational disorder as a consequence of the lack of metal ions.Therefore, a characterization of the apo state in solution of WTSOD1 and its mutants is necessary.

The 1H-15N HSQC spectra of the apo forms of both WT SOD1and the 2 pathological mutants i.e., T54R and I113T, havereduced signal dispersion with respect to those of the metalatedform (Fig. S1), indicating that some parts of the protein do nothave a well-defined conformation. High protein instability andstrong tendency to form high molecular weight oligomers (13)prevented us from collecting the triple resonance NMR exper-iments necessary to achieve a specific resonance assignment at298 K. A protein sample analysis, aiming at finding the bestcompromise between protein stability toward oligomerizationand line broadening effects of NMR signals, led us to acquire allof the spectra at 0.6 mM protein concentration and 288 K. Inthese experimental conditions, dimeric apo WT SOD1 is stablefor periods long enough to collect some of the triple resonanceNMR experiments necessary for sequence-specific resonanceassignment even if more than one sample from the samepreparation was necessary to acquire all of the experiments.

Through the NMR experiments, 68% of the backbone atoms(N, HN, and C�) were assigned for apo WT SOD1. Theunassigned peaks were all clustered in the central part of the1H-15N HSQC spectrum, thus experiencing severe resonanceoverlap. They were located mainly in loops connecting the�-strands, in particular in loop IV, which contains most of themetal-binding residues. This spectral pattern was already ob-served in the apo state of the monomeric form of SOD1 obtainedthrough residue mutations at the subunit–subunit interface (17).In the latter case, mutations of the 2 free cysteines (6 and 111)(as WT SOD1) prevented oligomerization, allowing to reach amuch higher protein concentration that, combined with the halfmolecular weight, led to a more complete assignment, which wasused here for comparison purposes.

From the analysis of the assigned chemical shift resonances itappears that the secondary structural elements of the �-sheet ofthe SOD1 �-barrel that comprises �-strands 1, 2, 3, and 6 existalso in the metal-free state, whereas �-strands 4, 5, 7 and 8 aremuch shorter. NH–NH long-range NOEs are indeed presentwithin �-strands 1, 2, 3, and 6, whereas they are mostly missingin the other �-sheet, which contains some of the metal ligandresidues (Fig. 2). From the analysis of the NOESY spectrum, italso appears that most of the long-range 1H–1H NOEs, involvingside-chain protons, are missing even within the secondary struc-tural elements.

Combined chemical-shift variations of backbone amidegroups between the dimeric apo SOD1 and its fully metallatedform (Fig. 3A) indicate significant structural changes in loop VII,similar to that already observed for monomeric apo SOD1 (17).Furthermore, a number of NH groups in this loop show, mainlyat low temperature, another set of signals with lower intensitycaused by a minor conformation or a group of fast exchangingconformers in slow exchange with the rest of the conformations.Analysis of the chemical-shift variations between the monomericand dimeric apo states of SOD1 (Fig. 3B) confirms that theabsence of metal ions similarly affects the 2 forms, particularlyin the electrostatic loop. The only detected differences can beascribed to the mutation of the 2 free cysteines residues (C6A

and C111S) and 3 residues (F50E, G51E, E133Q), which, byinducing protein monomerization, sizably affects the NMRsignals of the residues at the dimer interface. The latter 5mutations are present only in the ‘‘artificial’’ monomeric form.

Fig. 2. Secondary structural elements (red) based on the chemical shift indexanalysis for the apo WT SOD1 protein. Backbone long range NOEs (blue sticks)determined from 15N-edited NOESY spectra. The locations of the free cysteinesCys-6 and Cys-111 are represented by green and yellow spheres, respectively. Theoxidation state of SOD1 cysteine residues was also investigated through 13C 1DNMR spectra (Fig. S2).

Fig. 3. Chemical shift variations comparison of monomeric and dimericSOD1 states. (A) Combined chemical shift variations of backbone amideresonances between dimeric apo WT SOD1 and dimeric (Cu2,Zn2)-asWT SOD1.The positions where any of the structures under comparison are mutated arehighlighted in red. (B) Dimeric apo WT SOD1 and monomeric apo-asWT SOD1.The positions where any of the structures under comparison are mutated arehighlighted in red. The combined chemical-shift variations �avg(HN) werecalculated as [((�H)2 � (�N/5)2)/2]1/2, where �H and �N are chemical-shiftdifferences for 1H and 15N, respectively. The locations of the largest differ-ences (�� value of at least 0.3 ppm) observed are shown as black spheres on thecrystal structure of the apo WT SOD1 protein (3ECU); the residues mutated inany of the structures under comparison are represented as red spheres.

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Also the dynamical properties of apo WT dimeric SOD1 areaffected by the absence of the metals (Fig. 4). The 15N relaxationrates of backbone NHs, measured in the temperature range 288to 310 K, are consistent with the protein being essentially onlyin the dimeric state, with its tumbling rate increasing withdecreasing temperature. Residues located in the first �-sheet ofthe �-barrel have R1 and R2 relaxation rates and 15N{1H}–NOEsvalues characteristic of a structured protein (0.75 � 0.04 s�1,27.6 � 0.7 s�1, and 0.76 � 0.03, respectively at 298 K). Similaraverage values were found for the assigned residues of strands �7and �8 (Fig. 4), which indeed remain significantly structured, asalso confirmed by the presence of a NH–NH long-range NOEbetween �8 and �1 (Fig. 2).

The relaxation rates and 15N{1H}–NOEs, which are homoge-neous in the �-strand structures, are instead dramatically altered

in the electrostatic loop VII. The latter has, at 298 K, lower thanaverage R2 values, higher than average R1 values, and lower15N{1H}–NOEs, which become even negative at 310 K, indicat-ing that its residues experience motions faster than the overallprotein tumbling, i.e., faster than a nanosecond. Internal motionsin the subnanosecond time scale were also observed in loop VIIof monomeric apo SOD1 (17), confirming that the absence ofmetal ions similarly affects the dynamical properties of bothmonomeric and dimeric apo forms. The overall spectral features insolution for loop VII suggest that this region, and the nonassignedresidues located in the other loop regions, sample a wide range ofconformations that interconvert each other very fast.

15N relaxation rates of backbone NHs were also measured forapo T54R SOD1 (Fig. S3). The results are overall very similar to

Fig. 4. Dynamic properties of apo WT SOD1. (A–C) Backbone 15N(H) relaxation parameters and heteronuclear 1H-15N NOEs for apo WT SOD1 at 288 K (A), 298K (B), and 310 K (C). (D) Logarithm of protection factors for the backbone amide groups of the apo WT SOD1, log10(kint/kexp), where kint is the intrinsic exchangerate for the unprotected amide groups (34).

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the data obtained for apo WT SOD1 (Tables S2–S4), showingthat, also for this mutant, the mobility of loop VII is dramaticallyaffected by the lack of metal ions; as also confirmed by thenegative values of the 15N{1H}–NOEs. The similar mobilitybehavior of apo WT and T54R mutant is consistent with theirsimilar rates of oligomerization (14).

Unfortunately, the I113T mutant is too unstable, preventingthe acquisition of relaxation data at temperature �288 K. At thistemperature this mutant has overall the same dynamical prop-erties as WT and T54R SOD1, being essentially structured in�-sheet 1 and �-strand 8, but showing higher backbone dynamicsin loop VII where a sizable number of residues have negative15N{1H}–NOEs values even at 288K (Fig. S4 and Table S2). Atvariance with WT and T54R SOD1 mutant, in I113T someresidues located in other loop regions experience extensivedynamics on the subnanosecond time scale. In particular, someresidues of loop V (residues 90–94), which connects �5 and �6,show a peculiar drop of the 15N{1H}–NOEs and R2 values anda corresponding increase of R1 values consistent with fast localmotions (Table S2). Interestingly, �5 and �6 are located at theedge of the 2 sheets constituting the SOD1 barrel and arethought to be involved in amyloid fibril formation throughintermolecular aggregation with other edge strands in otherfALS-related mutants (18).

The observed local flexibility of the SOD1 apo forms withrespect to the metalated ones is consistent with their solventaccessibility. Indeed, the overall number of NH protons exchangingfast with the bulk solvent, as measured from H2O/D2O exchangeprocesses, is much higher in the apo state than in the metalated one.In the apo forms of both WT and T54R SOD1, after 20 min thesamples have been dissolved in D2O, �60 residues were completelyexchanged, whereas only �30 were exchanged in the metalated one.Nevertheless, hydrogen exchange protection factors of apo WT andT54R SOD1 (Fig. 4D and Fig. S3D) reveal that residues located inthe secondary structure elements of the �-sheet constituted bystrands �1, �2, �3, and �6 and some residues of �8 still show asignificant degree of order, in agreement with the presence of a�-structure hydrogen-bond pattern. In particular, in �6 it waspossible to quantify exchange rate values only for the amide protonsinvolved in hydrogen bonds with residues located in �3, whereas theother amide protons exchanged too fast with the solvent. OverallWT and T54R SOD1 have very similar H/D exchange properties.

Particularly relevant for the oligomerization process thatoccurs for apo WT SOD1 at physiological conditions (13), is thesolvent exposure of the free cysteines (Cys-6 and Cys-111).Although Cys-111 is highly solvent exposed in both proteinforms, Cys-6 has a dramatically different solvent accessibility(Fig. S5). In the metalated form its NH is essentially buried andprotected from the solvent and indeed its NH signal is stillpresent after 5 days in D2O. On the contrary, in the apo form italmost completely disappears after only 4 h. Also, the NH signalsof adjacent residues (residues 4–8, �-strand l) all disappear in4 h, suggesting a high solvent accessibility of the region aroundCys-6. The high solvent accessibility of Cys-6 and �-strand l isobserved for apo T54R mutant as well, suggesting, as for apo WTSOD1, that the metalated form is rigid and solvent protected,whereas the lack of metal ions makes this region and the entireprotein highly dynamic and more solvent accessible.

All of these features indicate that the apo state of SOD1 insolution is characterized by a distribution of conformations,particularly for �-strands 4 and 5 and loop VII.

Similar behavior was observed in the SOD-like protein fromBacillus subtilis (27), where its NMR properties indicate aconformational mobility for most of the protein, characterizedby defined secondary-structure elements and a dynamic tertiarystructure, at variance with the X-ray crystal structure of the sameprotein, which shows a well-ordered tertiary structure.

The overall studies here presented for the apo state of SOD1and its mutants in solution also explain the behavior of theseproteins with respect to crystallization. Crystallization trialswere performed at 2 different temperatures on both the apo andmetalated forms of WT SOD1 and the 2 mutants (Table S5), i.e.,at 288 K, which is the temperature at which the crystals discussedabove were obtained, and at 310 K, which is the temperature atwhich the oligomerization studies were carried out (13, 14).Crystals of the apo state of WT SOD1 and the 2 mutants can beobtained only at 288 K, whereas crystals for the metalated formsof WT SOD1 and the mutants were obtained at both temper-atures. This result indicates that temperature has a majorinfluence on the crystallization of the apo state whereas it isalmost negligible on the metalated one. This overall behavior isconsistent with the dynamic properties and conformationaldisorder of the apo state. A decrease in temperature slows downthe interconversion process among the various conformationsand increases the population of the most stable states, which cantherefore crystallize.

The local disordered state observed for the metal-free SOD1may appear to be in contradiction with its cooperative unfoldingbehavior reported by differential scanning calorimetry measure-ments (28). This contradiction can be, however, reconciled by thepresence of a significant portion of still existing tertiary structureand extensive structural interactions at the dimer interfacecaused by the preserved dimeric nature.

Concluding RemarksWe have recently shown (13) that oligomerization of apo SOD1involves oxidation of the 2 free cysteines (Cys6 and Cys111) withthe formation of intersubunit disulfide bonds, thus linking a highnumber of protein molecules in high molecular weight species.Some have suggested that the formation of SOD1 aggregates arethe consequence of both covalent disulfide cross-linking andnoncovalent interactions (29), whereas others proposed thatextensive disulfide cross-linking is not required for the formationof mutant SOD1 aggregates (30). Recent studies showed theimportance of nonphysiological intermolecular disulfide bondbetween cysteines 6 and 111 in mutant SOD1 for high molecularweight aggregate formation for protein ubiquitylation and neu-rotoxicity, which are all dramatically reduced when these cys-teines are substituted (31). In any case, there is a generalagreement on the critical role played by cysteines 6 and 111, inthe modulation of human SOD1 aggregation (29, 31).

We have shown that only the lack of metal ions makes SOD1oligomerization possible (13). The reason for the dramaticdifferent behavior of apo and metalated forms of SOD1 is nowbetter understood. Indeed, the solvent exposure of the reducedcysteines changes dramatically from the metallated form to theapo one. Only in the latter state a free cysteine can bind anotherone of a different monomer to form the soluble oligomer. Thecrystal structures, on the contrary, are not informative on thisrespect as they clearly represent only one of the multipleconformations taken in solution by the protein. Consistently, theapo form of both WT and the mutants fail to crystallize atphysiological temperature because of the high disorder andinternal mobility.

The information obtained from the NMR spectra indicatesthat in solution apo WT SOD1 samples a range of conforma-tions, which are highly disordered in some parts. Higher tem-peratures accelerate exchange among these conformations andcould populate new ones. This behavior explains why only thedisordered, locally unfolded, metal-free state has a dramaticprotein flexibility that makes accessible conformations prone tooligomerize, whereas the rigid structure of the metalated proteinis unable to do it.

Overall the present extensive structural and dynamical char-acterization of the apo state of WT SOD1 and some of its

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mutants showed that the lack of metal ions and the subsequentprotein flexibility allows the free cysteines (in particular Cys-6)to become exposed and therefore ready to get oxidized and formthe disulfide bonds that give rise to the soluble oligomers.

Materials and MethodsProtein Samples. Apo and metallated WT, T54R and I113T SOD1 samples wereexpressed and purified as described in SI Text.

Crystallization, Data Collection, and Structure Solution. Crystals of metal-freeSOD1 (WT, T54R, and I113T) were obtained by using the vapor diffusiontechnique at 288 K from 0.1 mM protein solutions containing 0.1 M Mes (pH6.5) or 0.1 M Hepes (pH 7.0), 20% PEG 3350. For further details on crystalli-zation, structure solution, and refinement see SI Text and Table S6.

NMR Experiments. NMR spectra were acquired at 288 K on an Avance 900Bruker spectrometer equipped with a cryogenically-cooled probe. Resonanceassignments of apo WT SOD1 form were performed through conventionalmultidimensional NMR techniques based on triple resonance experimentssummarized in Table S7. Because of the instability of the apo form of WT,T54R, and I113T SOD1, �1 sample was needed to complete NMR data collec-tion at 288 K. Samples of apo WT and T54R SOD1 were stable for �7 days at288 K, 5 days at 298 K, and only 3 days at 310 K, whereas I113T SOD1 mutantwas stable only for 3 days at 288 K.

The dynamic properties of the apo dimeric forms of apo WT, T54R, andI113T SOD1 were directly sampled through 15N relaxation measurements. 15Nlongitudinal and traverse relaxation rates and 15N{1H}–NOEs were recorded at288, 298, and 310 K for both WT and T54R mutant at 500 MHz, using a proteinconcentration of �0.6 mM. For I113T mutant it was possible to carry out 15Nrelaxation measurements only at 288 K because of its very high instability. R1

and R2 relaxation rates and heteronuclear NOE values were obtained asdescribed in Table S7.

For apo WT SOD1, the average backbone 15N longitudinal R1 and transver-

sal R2 relaxation rates and 15N{1H}–NOEs values, calculated excluding residues125–142, were 0.64 � 0.04, 34.0 � 1.6 s�1, and 0.70 � 0.03, respectively at 288K, 0.81 � 0.05, 27.3 � 1.2 s�1, and 0.72 � 0.03 at 298 K, and 1.15 � 0.06, 19.4 �0.9 s�1, and 0.75 � 0.03 at 310 K. For apo T54R SOD1, the values were 0.66 �0.05 s�1, 33.0 � 1.9 s�1, and 0.66 � 0.05, respectively at 288 K, 0.77 � 0.05,28.4 � 0.05, and 0.71 � 0.05 at 298 K, and 1.17 � 0.07, 20.1 � 1.0 s�1, and 0.73 �0.04 at 310 K. For I113T SOD1, the values were 0.71 � 0.05, 29.8 � 1.5 s�1, and0.60 � 0.06, respectively at 288 K .

A correlation time for protein tumbling (�c) of 22.6 � 1.9 ns at 298 K wasestimated from the R2/R1 ratio excluding those residues exhibiting below-average 15N{1H}–NOEs values and those experiencing conformational pro-cesses. The result is consistent with that obtained with the HYDRONMRprogram for apo SOD1 being only in a dimeric state.

H2O/D2O exchange properties were analyzed on samples obtained byrapidly diluting concentrated WT and T54R SOD1 both in metallated and apoforms at pH 7.0 [which was corrected for the isotope effect as described (32)],with D2O buffer to a final D2O/H2O ratio of 0.90. H/D exchange rates wereinvestigated through a series of 1H-15N SOFAST-HMQC experiments (33) per-formed from 20 min after dilution for 21 h every 9 min and then later acquiredafter 5 days. All experiments were carried out at 298 K. Exchange rates (kex)were determined by fitting the decay of the peaks intensities in the 1H-15NSOFAST-HMQC spectra as a function of time to a monoexponential decay(Fig. S6).

ACKNOWLEDGMENTS. We thank Marie-Paule Strub for providing the cys-teine-auxotrophic strain BL21(DE3)cysE. This work was supported by Euro-pean Commission ‘‘Understanding Protein Misfolding and Aggregation byNMR’’ (UPMAN) Grant LSHG-CT-2004-512052 (11/1/04-10/31/07), Ministerodell’Universita e della Ricerca– Fondo per Gli Investimenti della Ricerca di BaseGrant RBLA032ZM7 (12/09/05 -12/09/10), Ente Cassa di Risparmio di Firenze‘‘Relazione varianti proteiche strutturali-malattie genetiche’’ and ‘‘Basi mole-colari di patologie umane correlate a disfunzioni della catena respiratoria,’’and Marie Curie Host Fellowship for Early Stage Research Training MEST-CT-2004-504391.

1. Valentine JS, Doucette PA, Potter SZ (2005) Copper-zinc superoxide dismutase andamyotrophic lateral sclerosis. Annu Rev Biochem 74:563–593.

2. Bruijn LI, Miller TM, Cleveland DW (2004) Unraveling the mechanisms involved inmotor neuron degeneration in ALS. Annu Rev Neurosci 27:723–749.

3. Jonsson PA, et al. (2004) Minute quantities of misfolded mutant superoxide dis-mutase-1 cause amyotrophic lateral sclerosis. Brain 127:73–88.

4. Wang J, Xu G, Borchelt DR (2006) Mapping superoxide dismutase 1 domains ofnon-native interaction: Roles of intra- and intermolecular disulfide bonding in aggre-gation. J Neurochem 96:1277–1288.

5. Deng HX, et al. (2006) Conversion to the amyotrophic lateral sclerosis phenotype isassociated with intermolecular linked insoluble aggregates of SOD1 in mitochondria.Proc Natl Acad Sci USA 103:7142–7147.

6. Furukawa Y, Fu R, Deng HX, Siddique T, O’Halloran TV (2006) Disulfide cross-linkedprotein represents a significant fraction of ALS-associated Cu, Zn-superoxide dis-mutase aggregates in spinal cords of model mice. Proc Natl Acad Sci USA 103:7148–7153.

7. Pasinelli P, et al. (2004) Amyotrophic lateral sclerosis-associated SOD1 mutant proteinsbind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron 43:19–30.

8. Ohi T, Nabeshima K, Kato S, Yazawa S, Takechi S (2004) Familial amyotrophic lateralsclerosis with His46Arg mutation in Cu/Zn superoxide dismutase presenting character-istic clinical features and Lewy body-like hyaline inclusions. J Neurol Sci 225:19–25.

9. Ferri A, et al. (2006) Familial ALS-superoxide dismutases associate with mitochondriaand shift their redox potentials. Proc Natl Acad Sci USA 103:13860–13865.

10. Okado-Matsumoto A, Fridovich I (2001) Subcellular distribution of superoxide dis-mutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria. J Biol Chem 276:38388–38393.

11. Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001) A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembranespace of mitochondria. J Biol Chem 276:38084–38089.

12. Bertini I, Mangani S, Viezzoli MS (1998) in Advanced Inorganic Chemistry, ed Sykes AG(Academic, San Diego), pp 127–250.

13. Banci L, et al. (2007) Metal-free SOD1 forms amyloid-like oligomers: A possible generalmechanism for familial ALS. Proc Natl Acad Sci USA 104:11263–11267.

14. Banci L, et al. (2008) SOD1 and amyotrophic lateral sclerosis: Mutations and oligomer-ization. Plos ONE 3:e1677.

15. Arnesano F, et al. (2004) The unusually stable quaternary structure of human SOD1 iscontrolled by both metal occupancy and disulfide status. J Biol Chem 279:47998–48003.

16. Field LS, Furukawa Y, O’Halloran TV, Culotta VC (2003) Factors controlling the uptakeof yeast copper/zinc superoxide dismutase into mitochondria. J Biol Chem 278:28052–28059.

17. Banci L, Bertini I, Cramaro F, Del Conte R, Viezzoli MS (2003) Solution structure of ApoCu,Zn superoxide dismutase: Role of metal ions in protein folding. Biochemistry42:9543–9553.

18. Strange RW, et al. (2003) The structure of holo and metal-deficient wild-type humanCu,Zn superoxide dismutase and its relevance to familial amyothrophic lateral sclerosis.J Mol Biol 28:877–882.

19. Rakhit R, et al. (2004) Monomeric Cu,Zn-superoxide dismutase is a common misfoldingintermediate in the oxidation models of sporadic and familial amyotrophic lateralsclerosis. J Biol Chem 279:15499–15504.

20. Hough MA, et al. (2004) Dimer destabilization in superoxide dismutase may result indisease-causing properties: Structures of motor neuron disease mutants. Proc NatlAcad Sci USA 101:5976–5981.

21. Elam JS, et al. (2003) Amyloid-like filaments and water-filled nanotubes formed bySOD1 mutant proteins linked to familial ALS. Nat Struct Biol 10:461–467.

22. Hart PJ, et al. (1998) Subunit asymmetry in the three-dimensional structure of a humanCuZnSOD mutant found in familial amyotrophic lateral sclerosis. Protein Sci 7:545–555.

23. Cardoso RM, et al. (2002) Insights into Lou Gehrig’s disease from the structure andinstability of the A4V mutant of human Cu,Zn superoxide dismutase. J Mol Biol324:247–256.

24. DiDonato M, et al. (2003) ALS mutants of human superoxide dismutase form fibrousaggregates via framework destabilization. J Mol Biol 332:601–615.

25. Cao X, et al. (2008) Structures of the G85R variant of SOD1 in familial amyotrophiclateral sclerosis. J Biol Chem 283:16169–16177.

26. Serag AA, Altenbach C, Gingery M, Hubbell WL, Yeates TO (2002) Arrangement ofsubunits and ordering of �-strands in an amyloid sheet. Nat Struct Biol 9:734–739.

27. Banci L, et al. (2005) A prokaryotic superoxide dismutase paralog lacking two Culigands: From largely unstructured in solution to ordered in the crystal. Proc Natl AcadSci USA 102:7541–7546.

28. Rodriguez JA, et al. (2002) Familial amyotrophic lateral sclerosis-associated mutationsdecrease the thermal stability of distinctly metallated species of human copper-zincsuperoxide dismutase. J Biol Chem 277:15932–15937.

29. Cozzolino M, et al. (2008) Cysteine 111 affects aggregation and cytotoxicity of mutantCu,Zn-superoxide dismutase associated with familial amyotrophic lateral sclerosis.J Biol Chem 283:866–874.

30. Karch CM, Borchelt DR (2008) A limited role for disulfide cross-linking in the aggre-gation of mutant SOD1 linked to familial amyotrophic lateral sclerosis. J Biol Chem283:13528–13537.

31. Niwa J, et al. (2007) Disulfide bond mediates aggregation, toxicity, and ubiquitylationof familial amyotrophic lateral sclerosis-linked mutant SOD1. J Biol Chem 282:28087–28095.

32. Glasoe PK, Long FA (1960) Use of glass electrodes to measure acidities in deuteriumoxide. J Phys Chem 64:188–190.

33. Schanda P, Kupce E, Brutscher B (2005) SOFAST-HMQC experiments for recordingtwo-dimensional heteronuclear correlation spectra of proteins within a few seconds.J Am Chem Soc 33:199–211.

34. Bai YW, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptidegroup hydrogen exchange. Proteins Struct Funct Genet 17:75–86.

Banci et al. PNAS � April 28, 2009 � vol. 106 � no. 17 � 6985

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