Disease-associated Mutations at Copper Ligand HistidineResidues of Superoxide Dismutase 1 Diminish the Bindingof Copper and Compromise Dimer Stability*□S

Received for publication, May 10, 2006, and in revised form, August 31, 2006 Published, JBC Papers in Press, November 8, 2006, DOI 10.1074/jbc.M604503200

Jiou Wang‡1, Amy Caruano-Yzermans§1, Angela Rodriguez¶1, Jonathan P. Scheurmann¶1,Hilda H. Slunt‡�1, Xiaohang Cao¶, Jonathan Gitlin§, P. John Hart¶**‡‡, and David R. Borchelt‡�§§2

From the Departments of ‡Pathology and §§Neuroscience, Johns Hopkins University, Baltimore, Maryland 21205, the §EdwardMallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, the ¶Department ofBiochemistry, the **X-ray Crystallography Core Laboratory, and the ‡‡Geriatric Research, Education, and Clinical Center,Department of Veteran’s Affairs, South Texas Veterans Health Care System, The University of Texas San Antonio HealthSciences Center, San Antonio, Texas 78229, and the �Department of Neuroscience, Santa Fe Health Alzheimer’s DiseaseResearch Center, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutantsthat cause familial amyotrophic lateral sclerosis (FALS) haveheightened reactivity with �ONOO and H2O2 in vitro. Thisreactivity requires a copper ion bound in the active site and is asuggested mechanism of motor neuron injury. However, wehave found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands forcopper and which is inactive, develop motor neuron disease.Using a direct radioactive copper incorporation assay in trans-fected cells and the established tools of single crystal x-ray dif-fraction, we now demonstrate that this variant does not stablybind copper. We find that single mutations at copper ligands,including H46R, H48Q, and a quadruple mutant H46R/H48Q/H63G/H120G, also diminish the binding of radioactive copper.Further, using native polyacrylamide gel electrophoresis and ayeast two-hybrid assay, the binding of copper was found to berelated to the formation of the stable dimeric enzyme. Collec-tively, our data demonstrate a relationship between copper andassembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly producetoxic radicals via copper-mediated chemistry.

Amyotrophic lateral sclerosis (ALS),3 which is characterizedby progressive muscle weakness and motor neuron loss, pre-sents as both sporadic and familial (FALS) illness. A subset ofFALS cases is caused by missense mutations in the superoxidescavenging enzyme, Cu/Zn-superoxide dismutase 1 (SOD1) (1,2). To date, over 100 different point mutations, and �5 earlytermination mutations have been linked to FALS (www.alsod.org) (for reviews see Refs. 2–4). Early studies of FALS-SOD1 enzymes demonstrated that some mutants retain highlevels of activity and relatively long half lives (5). Moreover,mutant proteins that are inactive or short-lived do not exhibitevidence of dominant negative action with regard to the super-oxide-scavenging activity of enzyme derived from the normalallele (6). In transgenic mice, the hyperexpression of the G93Aand G37R variants of FALS-SOD1 increases superoxide scav-enging activity, kills motor neurons, and causes paralysis (7, 8).SOD1 knock-outmice do not develop ALS-like phenotypes butdo show sensory and motor neuropathy (9). Together, thesestudies establish that SOD1 mutations cause ALS through agained toxic property. Although expressed ubiquitously (8, 10),mutant SOD1 selectively damages motor neurons, by mecha-nisms yet to be fully understood.Each subunit of the mature homodimeric SOD1 enzyme

binds one atom of copper and one atom of zinc and contains asingle oxidized disulfide bond between Cys-57 and Cys-146(11–13). Because copper can participate in many types ofpotentially deleterious reactions, the role of the copper cofactorof SOD1 in the toxicity associatedwithmutant protein has beenintensely studied (for review see Refs. 4 and 14). To dissect therole of copper in the toxicity of mutant SOD1, we have exam-ined the impact of mutations of the four histidine residues thatare the primary copper ligands of the enzyme. Two of the fourhistidine residues that coordinate copper have been docu-mented as targets of natural FALSmutations;His-46 toArg andHis-48 to Gln (www.alsod.org). Both of these mutants arealmost completely devoid of superoxide-scavenging activity(15). Combining these two mutations into one protein pro-

* This work was supported by National Institutes of Health (NIH) GrantNIH44464 (to J. G.), by the ALS Association (to D. R. B.), by the MuscularDystrophy Association (to D. R. B.), by Robert A. Welch Foundation GrantAQ-1399 (to P. J. H.), by the Packard Center for ALS Research at JHU (toD. R. B.), and by NINDS, NIH Grants P01 NS049134 (to D. R. B. and P. J. H.)and R01 NS39912 (to P. J. H.). The production of 64Cu at the WashingtonUniversity School of Medicine is supported by NCI, NIH Grant R24 CA86307.The X-ray Crystallography Core Laboratory is supported by the VPR and theExecutive Research Council at the University of Texas Health Science Cen-ter at San Antonio. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental text, references, and Figs. S1 and S2.

The atomic coordinates and structure factors (code 2NNX) have been depositedin the Protein Data Bank, Research Collaboratory for Structural Bioinformat-ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Neuroscience, Uni-

versity of Florida, 100 Newell Drive, Room L1–100H, P. O. Box 100244,Gainesville, FL 32610 – 0244. Tel.: 352-294-0105; Fax: 352-392-8347; E-mail:Borchelt@mbi.ufl.edu.

3 The abbreviations used are: ALS, amyotrophic lateral sclerosis; FALS, familialALS; SOD1, Cu/Zn-superoxide dismutase 1; CHO, Chinese hamster ovary;Quad, quadruple mutant H46R/H48Q/H63G/H120G.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 1, pp. 345–352, January 5, 2007Printed in the U.S.A.

JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 345

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

duces a molecule that also lacks demonstrable activity butretains high toxicity to motor neurons (16). Additional substi-tutions at the two other copper ligands (His-63 to Gly and His-120 to Gly), eliminate the copper-binding ligands, generating aprotein that remains capable of inducing motor neuron diseasein transgenic mice (17). Studies of SOD1-H46R suggest thatthis single mutation interferes with copper binding (18, 19),whereas the H48Q mutant can be made to bind copper in thecorrect site, although with an altered coordination geometry(20). The copper-binding abilities of H46R/H48Q or SOD1-Quad are also predicted to be severely compromised, but thishas not yet been demonstrated experimentally.To fill this gap in knowledge, we here study copper binding of

four SOD1 variants, H46R, H48Q, H46R/H48Q, and H46R/H48Q/H63G/H120G (Quad), using a direct radioactive copperincorporation assay. In transfected cell models, we show thatnone of these variants possess high affinity for copper. We alsouse single crystal x-ray diffraction to examine directly the cop-per binding site of SOD1-H46R/H48Q protein expressed inyeast and isolated by standard non-denaturing biochemicalmethods. This analysis reveals that the H46R/H48Q mutantprotein does not bind copper in either of the metal-bindingsites of the protein. We also noted that mutation of copperligands correlated with reduced ability to form stable dimers,using native gel electrophoresis and a yeast two-hybrid assay.We interpret these findings as evidence that the loss of copper-binding His ligands in SOD1 reduces the stable binding of cop-per, and that the lack of such bindingmay underlie the inabilityof these mutants to mature into stable dimeric enzymes. Wenote that nearly all of SOD1-H46R/H48Q or SOD1-Quad pro-teins found in spinal cord tissues display an electrophoreticmigration pattern of monomeric enzyme, suggesting that thepoor incorporation of copper in these mutants also occurs inspinal cord tissues.

EXPERIMENTAL PROCEDURES

Expression Plasmids—cDNA of human SOD1 harboring thefollowing mutations, A4V, G37R, G85R, G93A, I113T, H46R,H48Q, H46R/H48Q, and Quad were inserted into the pEF.Bosvector. Each of these plasmids has been described and used inprior studies (5, 6, 15–17).Copper Incorporation Studies—The general methods used in

metabolic radiolabeling with 64Cu have been described previ-ously (21). Briefly, 64CuCl2 was obtained from Michael Welchat Washington University School of Medicine with a specificactivity of 50–200 mCi/�g. CHO cells were transfected 48 hprior to copper metabolic labeling with 5 �g of DNA of theconstructs noted using Lipofectamine 2000 (Invitrogen). Cellswere labeled in Opti-Mem (Invitrogen) containing 50 �Ci/ml64Cu for 3 h at 37 °C. Cells were washed, harvested, and lysed inNonidet P-40 lysis buffer (50 mM HEPES, 250 mM NaCl, 0.1%Nonidet P-40, 5 mM EDTA, pH 7.6) containing protease inhib-itors. The lysate, 100 �g, was mixed with Laemmli samplebuffer (final SDS concentration 1%) (22) and electrophoresedon a non-reducing 10% polyacrylamide gel containing 0.1%SDS. Samples were not heated prior to electrophoresis. The gelwas exposed to PhosphorImager plates (Amersham Bio-sciences) overnight; plates were analyzed in the instrument as

described by the manufacturer. For SOD1 immunoblot, the gelfrom the copper labeling study was transferred at 400 mA for1 h then blocked in 5%nonfatmilk in phosphate-buffered salinewith Tween-20 (standard protocol, Pierce Biotechnology). Pri-mary �-SOD1 antibody (SOD1 100, Stressgen BioreagentsCorp.-Nventa Biopharmaceuticals Corp., San Diego, CA) at1:5,000was incubated overnight at 4 °C. The blot waswashed inphosphate-buffered saline with Tween-20 followed by second-ary antibody (goat �-rabbit-horseradish peroxidase, Pierce) at1:10,000 and washed prior to development with the Pico Kit(Pierce).Yeast Two-hybrid Assessment of SOD1 Dimer Formation—To

assess howFALSmutations effect homodimeric interactions, weutilized a yeast two-hybrid assay to measure interactionsbetween mutant and wild-type subunits. The variants testedincluded the following: the G37R, G93A, and I113Tmutations,which have been previously established to form stable dimers(5, 6, 23); the H46R, H48Q, H46R/H48Q double mutant, andthe Quad variants, which affect residues critical for the coordi-nated binding of copper (13); and the A4V mutant, which hasbeen suggested to be prone to monomerization (24). The A4V,G37R, G85R, G93A, H46R/H48Q, and Quad variants have allbeen expressed in transgenic mice to producemousemodels ofFALS (7, 8, 16, 25–27).The procedure for developing the assay involved the follow-

ing, using the DupLEX-A system (OriGene Technologies,Rockville, MD). First, each of the 10 different mutants, and thewild-type cDNAs, were fused to the LexA DNA-bindingdomain of the bait-fusion protein (plasmid pEG202 betweenEcoR1 and BamH1 sites). Wild-type SOD1 cDNA was clonedinto the target plasmid pJG4–5 between EcoR1 and XhoI siteslocated at 3� of the activation domain. Bait plasmidswere trans-formed into the EGY48 strain of yeasts, and the target plasmidwas transformed into themating strain RFY206. Beforemating,yeasts harboring the bait constructs were screened for suffi-cient expression of the bait-fusion protein to repress transcrip-tion of a reporter gene in the plasmid pJK101 encoding �-ga-lactosidase under the transcriptional control of a constitutivepromoter interrupted by the LexA operator. Yeast cells werecultured on media containing 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-gal, Sigma-Aldrich). For each bait con-struct, we identified three or four independent yeast clones thatwere white; indicating complete repression of �-galactosidaseexpression. Low expression of �-galactosidase in each colonywas verified by liquid assay. This approach established that eachclone expressed the bait-fusion protein at levels sufficient tosaturate the DNA-binding site recognized by the LexA domainof the fusion protein.Each of the three clones harboring the bait-fusion and

reporter constructs were then made ready for mating to theRFY206 yeast strain (harboring the SOD1-wt target fusion pro-teins) by culturing the yeast onmedia containing 5-fluorooroticacid (Sigma-Aldrich). The pJK101 repressor-reporter plasmidsutilize URA-3 as the selectable marker gene, allowing the use ofmedia containing 5-fluoroorotic acid to force the segregation ofthe bait-fusion constructs and the reporter construct (URA-3 isan enzyme in the uracil biosynthetic assay, which acts upon5-fluoroorotic acid to produce a toxin). Only cells that have lost

Role of Copper in SOD1 Dimerization and Neurotoxicity

346 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 1 • JANUARY 5, 2007

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

the repressor-reporter construct will grow. Again, three inde-pendent colonies were isolated, and these clones were screenedby growth on media lacking uracil to ensure loss of pJK101plasmid. These strains were then mated to SOD1-wt/RFY206yeast that also harbored a plasmid (pSH18–34) encoding �-ga-lactosidase behind a promoterless LexADNA-binding domain.Initially, yeast cells harboring all three constructs were selectedsolely on the basis of selectable-marker genes within the plas-mids. Again, three independent clones were isolated and cul-tures of yeast harboring both genes were then grown for lysisand liquid assay of �-galactosidase activity. Yeast cells werelysed mechanically, and the total amount of protein in eachlysate was measured by a BCA kit (Pierce). The liquid assaycontained a colorimetric substrate, 2-nitrophenyl-�-D-galacto-pyranoside, at a concentration of 0.67 mg/ml, and lysate from�1 � 106 cells or 0.2 A600 units. Reactions were incubated at28 °C for 15min before absorbancewas read byA420 on a stand-ard spectrophotometer (Amersham Biosciences).Structure Determination of SOD1-H46R/H48Q—TheH46R/

H48Q double mutant protein was expressed in yeast, purified,and characterized as previously described (28) with the addi-tion of a DEAE-Sephadex chromatography step between thehydrophobic interaction chromatography and gel-filtrationcolumn steps. The metal content of the double mutant prior tocrystallization screens was determined using inductively cou-pled plasma-mass spectrometry. Crystals suitable for x-ray dif-fraction work were grown by the hanging drop, vapor-diffusionmethod. H46R/H48Q SOD1 at 26mg/ml in 2.25mMpotassiumphosphate, pH7.0, wasmixedwith a precipitating solution con-sisting of 20% polyethylene glycol 1000, 0.1 M phosphate, pH6.2, 0.2 M NaCl at 25 °C. Hexagonal plates appeared within 2months. A suitable specimenwas flash-cooled in liquid nitro-

gen using mother liquor made 20%(w/v) in polyethylene glycol 400 asa cryoprotectant. Diffraction datawere gathered at beam line 4.2.2at the Advanced Light Source,Berkeley, CA. All diffraction datawere processed using the programd*TREK (29).The structure was determined by

molecular replacement using theprogram MOLREP (30). A mono-mer of human G37R SOD1 (31) wasused as the search model. Crystallo-graphic refinement was performedinitially in CNS (32) and, in laterstages, in SHELX-97 (33). The pro-gramCOOT (34) was used forman-ual adjustment of the molecularmodels.

RESULTS

Previous studies had establishedthat natural pathogenic mutationsat histidines 46 (H46R) and 48(H48Q) drastically reduce enzymeactivity (15). Not surprisingly, com-

bining the H46R/H48Q mutations into one variant or addingexperimental mutations H63G/H120G did not restore activity(16, 17). However, whether any of these mutants stably bindcopper has not been directly assayed.In previous work, we demonstrated that SOD1-H46R/H48Q

is relatively stable, inactive, and capable of inducingmotor neu-ron disease in transgenic mice (16). To examine the copper-binding site of thismutant, purified proteinwas crystallized andanalyzed as described under “Experimental Procedures.” Anal-ysis of the copper- and zinc-binding sites of the double mutantsuperimposed on SIGMAA electron density contoured at 1.5 �demonstrated that the mutations preclude copper ion bindingat the copper site without spurious binding in the zinc site (Fig.1). This observation agrees with data obtained from inductivelycoupled plasma-mass spectrometry on the protein sample priorto crystallization, which returned values of 0.05 equivalents ofcopper and 1.5 equivalents of zinc, per dimer.To assess copper binding by these variants, and others, in

mammalian cells, we used a cell transfection model to expresshigh levels of mutant SOD1 in CHO cells. 48 h after transfec-tion, cells were incubated with 64Cu, then lysed in buffers withnon-ionic detergent and chromatographed on non-reducing,non-denaturing 10% polyacrylamide gels, containing 0.1% SDSand standard Laemmli buffers (22), before exposure to Phosphor-Imager plates (21). Cells transfected with human SOD1-wtexpression plasmids contained abundant levels of radiolabeledhomodimer enzyme, whereas in cells transfected with theH46R, H48Q, H46R/H48Q, or Quad variants, the only labeledprotein evident was the endogenous CHO protein (Fig. 2A).Because these gels allow for assay of crude cell lysates andbecause there are currently no radioactively labeled forms ofzinc that are available to us, whether any of the proteins visual-

FIGURE 1. The metal-binding sites of the human FALS SOD1 double mutant H46R/H48Q (left) and thewild-type enzyme (right). The structure surrounding the copper-binding site of one subunit of SOD1-H46R/H48Q is compared with SOD1-wt (right). All subunits in the crystal of SOD1-H46R/H48Q showed perturbedcopper-binding sites. The structure shown is that of the crystal subunits that most closely resemble SOD1-wt.Arg-143 and residues corresponding to metal ions ligands in the wild-type enzyme (metal ligand 46, 48, 63, 71,80, 83, and 120) are labeled. The metal ions are represented by spheres. Metal ligand and hydrogen bonds areshown as dotted lines. In the left image, the side chain of the Arg residue substituted at position 46 donates ahydrogen bond to the carbonyl oxygen of Thr-137 on the opposite side of the active site channel, preventingthe binding of copper ion (see text).

Role of Copper in SOD1 Dimerization and Neurotoxicity

JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 347

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

ized on these gels contain zinc could not be determined. Thusapo/holo designation refers to the absence/presence of copperonly, respectively, here and throughout this report.To confirm expression of the mutant proteins and to deter-

mine the relative position of migration in these gels, after expo-sure to the PhosphorImager plates, the gels were electrotrans-ferred to nitrocellulose for immunoblotting as described under“Experimental Procedures.” The only SOD1 found in thelysates of cells transfected with the histidine mutants displayedan electrophoretic migration consistent with the previouslyreported electrophoretic migration of monomeric SOD1 (21,35), whichmigrates much faster than the holo, dimeric enzyme(also see supplemental Fig. S2). Note the lack of 64Cu labeling(in Fig. 2A) at positions on the gel corresponding to the positionof the four histidine variants (Fig. 2B).To further address the effect of FALS mutations on dimer

interactions, we developed a yeast two-hybrid assay tomeasureinteractions between mutant and wild-type subunits. The vari-ants tested included the four histidine variants described aboveas well as the A4Vmutant, which has been reported to be proneto monomerization (24), and the G37R, G85R, G93A, and

I113T mutants, which we have previously established to formstable active dimers (5, 6, 23, 36). The assay involved two stepsdescribed in detail under “Experimental Procedures.” In thefirst step, yeast cells were transfected with plasmids that harborthe bait fusion constructs (each mutant cDNA fused to a LexADNA-binding domain) and a reporter plasmid to assess thestable expression of the bait-fusion protein. Three independentyeast clones showing complete repression of reporter tran-scription were isolated for each bait-fusion construct. Thisassay established that the expression of the various bait proteinswas at a level sufficient to saturate binding of the promoterelements of reporter constructs. Each of these clones was thencultured in selection media to screen for segregation of thebait-fusion constructs and the repression-reporter construct.Three independent clones for each were isolated and culturedinmedia to confirm loss of the reporter construct. In the secondstep, these strains were mated to yeast harboring the target-fusion construct (SOD1-wt/transcriptional activator domain)and a promoterless �-galactosidase reporter plasmid beforeselection solely on the basis of selectable-marker genes withinthe plasmids. Assays for �-galactosidase activity in yeast lysatesrevealed significant variation in the strength of bait-targetfusion interaction to promote �-galactosidase production (Fig.3). The wt, G37R, G85R, G93A, and I113T bait-fusion con-structs showed strong activation of �-galactosidase synthesis.Whereas, the A4V construct, and all variants harboring muta-tions at copper-binding histidines, showed very poor activationof �-galactosidase production (Fig. 3).Given the apparent correlation between mutation of copper

ligand residues and loss of stable homodimer interactions, weprepared extracts of spinal cords from transgenic mice thatexpress wt, G37R, G85R, G93A, H46R/H48Q, and Quad vari-ants of human SOD1. These extracts were then electrophore-

FIGURE 2. FALS mutations at copper-binding histidine residues of SOD1dramatically reduce affinity for copper. A, CHO cells were transfected toexpress human SOD variants before being metabolically labeled with 50�Ci/ml of 64Cu for 3 h. 100 �g of each cell lysate was separated on a non-reducing 10% polyacrylamide gel containing 0.1% SDS. The 64Cu autoradio-gram shows Cu-labeled endogenous hamster SOD1 dimer (solid arrow) in allsamples, and Cu-labeled human SOD1 dimer (solid arrowhead) only in the WTsample. B, a duplicate of the gel used for 64Cu autoradiogram was analyzed bySOD1 immunoblot, which reveals endogenous hamster SOD1 monomer andhuman SOD1 monomers that are not labeled by 64Cu. Note: apo refers to theabsence or presence of copper.

FIGURE 3. FALS mutations at copper-binding histidine residues of SOD1 dra-matically reduce the strength of normal dimer interactions. Interactionsbetween the human WT SOD1 and mutant subunits were measured by a yeasttwo-hybrid assay (see “Experimental Procedures”). Yeasts expressing mutant-SOD1 bait-fusion proteins were mated with yeasts expressing wild-type SOD1target-fusion proteins. The interactions between two subunits were recorded byinduction of a �-galactosidase reporter gene. Enzyme activity was measured byoptical density values per microgram of yeast protein extract and then normal-ized to the I113T, which had the highest levels of�-galactosidase production. Thedata represent means�S.E. Three or four independent bait fusion yeast colonies,all of which express sufficient proteins to pass a repression assay, were used foreach SOD1 variant (total number of measurements for each variant, n � 9–12).Significance levels were determined by one-way analysis of variance with Bon-ferroni corrections; the asterisks mark interaction measurements that are signifi-cantly lower than that of the wild-type to wild-type interaction values andmutant to wild-type interaction values with p � 0.01.

Role of Copper in SOD1 Dimerization and Neurotoxicity

348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 1 • JANUARY 5, 2007

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

sed on native gels for SOD1 activity assay (5, 37) or were trans-ferred to nitrocellulose and immunoblotted with a humanSOD1-specific antiserum (Fig. 4, A and C). To determine thelevels of SOD1 in each sample, fractions were also mixed with2� Laemmli buffer and analyzed by standard SDS-PAGEand immunoblot with an antiserum that detects both mouseand human SOD1 (Fig. 4B). In the extracts from wt, G37R, andG93A mice, the pattern of activity in the assay gel (Fig. 4A)matches well with the pattern of immunoreactivity on theimmunoblot of the native gel (Fig. 4C). However, for miceexpressing H46R/H48Q and SOD1-Quad, there is no evidenceof transgene-derived superoxide-scavenging activity (Fig. 4A),

and the migration pattern of these proteins (Fig. 4C) resemblesthat of wild-type enzyme reduced in the presence of chelatingagents (see supplemental Fig. S2). A small amount of thesemutants co-migrated with SOD1-wt and SOD1-G93Ahomodimers and may therefore be in a homodimeric state.Clearly, however, the majority migrates much more rapidly,similar to metal-deficient/reduced wild-type protein.Analysis of extracts from the mice expressing the G85R var-

iant reveal data consistent with the yeast two-hybrid assay inthat G85R SOD1 is capable of forming a dimeric protein (Fig.4C). G85R is reported to retain partial activity in a solutionassay, but its activity is undetectable in the gel assay (15). It ispossible that copper was initially loaded in the dimeric enzymeand then lost during some part of the processing and gel elec-trophoresis. Alternatively, the G85R variant may have copperbound, stabilizing the homodimer, but for some other reason isless able to catalyze superoxide disproportionation in the gelassay.

DISCUSSION

In previous work, we established that transgenic miceexpressing SOD1-H46R/H48Q develop motor neuron diseasetypical of FALS (16, 17). Although this mutant lacks detectablesuperoxide disproportionation activity (16), whether thismutant stably binds copper was not known. Here, we providethree lines of evidence that indicate that this protein does notstably bind copper. First, the crystal structure of SOD1-H46R/H48Qpurified fromyeast indicates an absence of copper ions inthe copper or zinc sites and substantial rearrangement of thecopper-binding pocket. Expression of SOD1-wt in the samesystem yields correctly metallated, active, homodimericenzyme (28). Second, inductively coupled plasma-mass spec-trometry analysis of the purified protein isolated from yeastindicates �0.05 equivalent of copper per unit of purified pro-tein (prior to crystallization). Third, cells transfected withexpression plasmids for SOD1-H46R/H48Q demonstrate littleor no 64Cu associated with this protein as assayed by autora-diography of non-reducing, non-denaturing gels. Together,these data establish that SOD1-H46R/H48Q does not possess ahigh affinity for copper.Moreover, we extend our analyses to other SOD1 variants

harboring mutations at Cu-ligand histidine residues. We findthat single mutations at H46R or H48Q, when expressed inCHOcells, also diminish the binding of radioactive copper. Notsurprisingly, the experimental mutant SOD1-Quad also lacksevidence of copper binding.We also find an additional propertyshared by SOD1 variants with mutations at His Cu-ligands. Innative gels, SOD1 variants expressed inCHOcells that encodedmutations at histidine ligands showed electrophoretic mobili-ties similar to that of the monomeric protein. In addition, themajority of mutant SOD1 in spinal cord extracts from miceexpressing both the H46R/H48Q and Quad mutants migratesat a position resemblingmonomeric protein.We therefore con-clude that SOD1-H46R/H48Q and SOD1-Quad have greatlydiminished ability to stably bind copper, which appears to alsoaffect the formation of the normal dimeric enzyme.Metal Binding and Dimerization of Mutant SOD1—The

absence of copper in the copper-binding site of the H46R/

FIGURE 4. The electrophoretic migration of mutant SOD1 (H46R/H48Qand Quad-His) isolated from spinal cord resembles that of metal-defi-cient/reduced wild-type SOD1. Spinal cords from pre-symptomatic trans-genic animals (2–3 months of age) were removed and homogenized in PBSby probe sonication for 30 s at 50% output (70 watts, Tekmar, Cincinnati, OH)and centrifuged at 100,000 � g for 5 min in a Beckman Airfuge (BeckmanCoulter, Inc., Fullerton, CA). A, 100 �g of supernatant protein was separatedby native gel electrophoresis and assayed for superoxide dismutase activityby gel assay as described under “Experimental Procedures.” Amounts (0.125–2.0 �g) of purified human SOD1 proteins were used as standards. Onlydimeric holoenzymes show activity. In contrast to the WT, G37R, and G93Ahuman SOD1 that show abundant active human holoenzymes (hDimer, solidarrow), H46R/H48Q, Quad, and G85R proteins show neither detectable activ-ity nor affect the migration of mouse SOD1 homodimer (mDimer, open arrow),confirming their inability to form heterodimers (m/hDimer, solid arrowhead).NTg � non-transgenic sample. B, 0.5 �g of supernatant protein from mousespinal cords was assayed for SOD1 protein levels by the standard SDS-PAGEusing an antiserum against a conserved region in human SOD1 (hSOD1, solidarrow) and mouse SOD1 protein (mSOD1, open arrow). Note: G85R humanprotein runs slightly above the mouse SOD1. C, supernatant protein (5 �g)from mouse spinal cords was separated by native gel electrophoresis andimmunoblotted using an antiserum that recognizes the human SOD1 but notthe mouse protein. The homodimers (solid arrow) formed by WT, G37R, orG93A are consistent with those in A. The majorities of H46R/H48Q and Quadmutants migrate at positions similar to reduced and de-metallatedhSOD1-WT (open arrow). The less abundant mutant G85R appears to migrateat a position expected for dimeric enzyme.

Role of Copper in SOD1 Dimerization and Neurotoxicity

JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 349

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

H48Qmutant is consistent with what is observed in two differ-ent crystal structures of singly substituted H46R SOD1 thatreside in the protein data bank, which contains a combinedtotal of twelve H46R SOD1 subunits (19, 20). In each case, theside chain of Arg-46 in these subunits donates a hydrogen bondto an acceptor across the active site channel. These hydrogenbond acceptors include the indole nitrogen of His-63, the car-bonyl or side-chain oxygen of Thr-137 (as in Fig. 1), or a side-chain oxygen of Asp-124 (19, 20). Taken together, these datasuggest that the H46R substitution alonemarkedly disrupts thebinding of copper in the copper site.In contrast, the singly substituted human H48Q SOD1 pro-

tein has been shown to bind copper ions at the copper site whenexpressed in the presence of high levels of copper (38) or whenre-folded in vitro (20). Spectroscopic analysis of SOD1-H48Q,expressed in insect cells grown in media supplemented withcopper and zinc sulfate (up to 300 �M), suggested that the cop-per is coordinated in a geometry that deviates from the dis-torted tetragonal arrangement found in wild-type toward onethat ismore regular (38). The coordination of copper ion boundto yeastH48QSOD1 reconstitutedwith two equivalents of cop-per and zinc per dimerwas observed directly in the x-ray crystalstructure refined to high resolution (PDB code 1F1A). In thiscase, the copper coordination geometry was found to be squarepyramidal, with a water molecule and the indole nitrogens ofHis-46, His-63, and His-120 in the square plane and an axialwater molecule acting as a fifth ligand. Thus, all of the studiesmentioned above provide evidence thatH48QSOD1 is capable ofbindingmetal ions in thecoppersite inanon-nativeconformation.Our cell culture labeling studies are generally in agreement

with the structural data in that SOD1 proteins with the H46Rmutation fail to show binding of radiolabeled copper. In con-trast to the studies cited above that used purified protein, wefind that the H48Q variant, when expressed in CHO cells, doesnot bind radiolabeled copperwith significant affinity. The load-ing buffers and gels used in 64Cu labeling experiments con-tained SDS at concentrations that do not affect the binding ofcopper to wild-type SOD1 (21). Although we cannot rule outthe possibility that the SDS removed loosely bound copperfrom the H48Q mutant, the electrophoretic migration ofSOD1-H48Q in these gels was identical to that of the other histi-dinemutants (seeFig. 2),which together are similar tomonomericwild-type protein (see below and supplemental Fig. S2).In yeast and human SOD1, the oxidation of the intrasubunit

disulfide bond between Cys-57 and Cys-146 stabilizes themonomeric subunit structure, facilitating the dimerization ofenzyme. This oxidation of the disulfide bond appears to bedependent upon the loading of copper (39, 40).When analyzingpurifiedwild-type human enzyme that has an oxidized disulfideand zinc bound in the zinc site, the removal of copper alone isnot sufficient to dissociate homodimeric enzyme (28). To pro-ducemonomers, removal of both themetal ions and the reduc-tion of the normal intramolecular disulfide bond are required(28). Therefore, for wild-type SOD1, monomerization is asso-ciated with both loss of metal and reduction of the disulfide.However, for the mutants we study here, we do not knowwhether monomerization also requires loss of the disulfidebond, although it seems likely. One simple interpretation of the

data would be that the His mutants monomerize, because theybind copper poorly. This interferes with the maturation of theprotein to generate structures that allow for normal dimericinteractions (39).Assays of SOD1 Dimerization—Native gel electrophoresis

has routinely been used to distinguish dimeric and monomericspecies of SOD1, with monomeric enzyme running signifi-cantly faster than dimeric enzyme (21, 35). A potential caveat inusing native gels is that some FALS mutations can affect theelectrophoretic migration of dimeric SOD1 (5). For example,SOD1-G37R dimers migrate more slowly than SOD1-wt (seeFigs. 4 and 5), whereas SOD1-G41Dmigrates more rapidly (5).In the analysis of protein expressed inCHOcells (see Fig. 2), thesample and gel buffers contained SDS, which masks the smallcharge effects of amino acid substitutions on gelmigration (21).In the study of proteins extracted from mouse tissues, nativegels lacking SDS were used, where charge can have a greatereffect on electrophoretic migration. However, the most robusteffect would occur by the substitution of His-46 for Arg (pI ofArg is 11.15 versus 7.41 for His), which should slow, rather thanspeed migration as it occurs for the G37R substitution (5).Moreover, other studies of SOD1-H46R purified from Sf9insect cells and analyzed by native gel (no SDS) also reportedmigration consistent with monomeric protein (38). Overall, webelieve that the histidine mutants are less able to form stabledimers.Whether the monomeric forms of these mutants bind zinc is

unknown. The crystals of SOD1-H46R/H48Q contained formsof the protein that resembled normal dimers and containedzinc bound correctly in the zinc site (see Fig. 1 and supplemen-tal Fig. S1). However, the role of zinc binding in the maturationof the protein in cell cytosol is uncertain and requires furtherstudy.The yeast two-hybrid assay we developed to assess dimer

interactions provides additional experimental validation of therole Cu-ligand residues have in dimer formation. As describedin under both “Experimental Procedures” and “Results,” wedemonstrated that the bait fusion proteins of eachmutant wereexpressed to sufficient levels to saturate the DNA-bindingdomains of reporter constructs. Thus, the lack of production of�-galactosidase is indicative of poor interaction with the targetSOD1-wt fusion protein. The bait fusions of SOD1-wt (positivecontrol) and three of the FALS mutant enzymes behaved aspredicted by other studies. Studies by us (5) and others (7, 23,36) have collectively established that the G85R, G93A, andI113T mutants can form stable dimers. We also note that theelectrophoretic migration pattern of G85R protein extractedfrom mouse tissues was consistent with dimeric enzyme (seeFig. 4C). The A4V variant did not interact with wild-type sub-units efficiently in the two-hybrid assay; data consistent withprevious reports that the A4V variantmonomerizes at low con-centrations (24). Moreover, other studies have demonstratedthat if A4V fails to bind copper and oxidize the intramoleculardisulfide, then the protein is essentially an unfolded monomer(41). Therefore, the data on SOD1-wt, SOD1-A4V, SOD1-G85R, SOD1-G93A, and SOD1-I113T in the yeast two-hybridassay are consistent with other biochemical data on thesemutants. The histidine mutants uniformly show very poor

Role of Copper in SOD1 Dimerization and Neurotoxicity

350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 1 • JANUARY 5, 2007

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

interactions in the two-hybrid assay, data that are corroboratedby the electrophoreticmigration of thesemutants in native gels.Notably, in the high protein concentrations used in crystalliza-tion, some of the H46R/H48Q subunits formed dimers resem-bling the mature enzyme (PDB accession code 2NNX). How-ever, at the concentrations present in cell cytosol or transgenicmouse tissues, our data indicate that themajority of the proteinis monomeric.We interpret these data as an indication that thefour variants we have analyzed here, harboring mutations atCu-ligand His residues, fail to adopt a structure compatiblewith the formation of stable dimers.In yeast, human SOD1 acquires copper via interactions with

the yeast copper chaperone for SOD1 or from reduced gluta-thione (42), and we therefore expect that the fusion proteinsused in the yeast two-hybrid experiments could acquire copper.Unfortunately, we found that the levels of SOD1-fusion proteinexpression in the yeast strains used in our assay were too low todetermine whether any of the proteins bound copper (i.e. wereactive in assay gels, data now shown). We therefore cannot becertain that the binding of copper by the wt, G93A, G85R, orI113T bait-fusion proteins is responsible for the ability to inter-act with SOD1-wt target-fusion proteins or that the lack ofcopper binding by the His mutants is responsible for the inabil-ity to for these bait-fusion proteins to interact with SOD1-wt.Because the Cu-ligand residues are not known to be directlyinvolved in bonding at the dimer interface (13, 43), we believe itlikely that poor binding of copper by the fusion proteins is onefactor in determining the quaternary structure and the ability toof subunits to dimerize.

CONCLUSIONS

In summary, we demonstrate that mutations at histidine res-idues critical for the coordinated binding of copper, particularlyH46R, dramatically reduce the ability of these enzymes to stablybind copper, which affects subsequent post-translational fold-ing to achieve dimeric structure. It is of interest that previousstudies have associated the H46R mutation in humans with avery slowly progressing form of the disease (44, 45), whichcould be construed as consistent with the idea that Cu-medi-ated toxicity plays a role in disease, because this mutant wouldbind copper poorly and thus be less toxic. However, in trans-genic mouse models, the dose of protein required for Cu-defi-cient variants to induce disease is similar to that of variants thatstably bind copper (16, 17). To explain this observation,mutants that bind copper weakly would have to somehow pos-sess toxicity equivalent to mutants that bind copper far better,and presumably are far more active in toxic Cu-mediatedchemistry. The simplest interpretation in our view is that Cu-mediated chemistry, when possible to occur, plays a secondaryrole in disease pathogenesis with othermechanisms driving thebasic disease process. Based on the observation that all miceexpressing mutant forms of SOD1, including the mutantsexamined here and very unstable C-terminal truncationmutants, accumulate aggregated species of mutant protein assymptoms progress (17, 46–48), we believe that aggregation ofthemutant protein is likely to be one of the centralmechanismsof toxicity.

REFERENCES1. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati,

A., Donaldson, D., Goto, J., O’Regan, J. P., Deng, H.-X., Rahmani, Z., Kri-zus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S. M., Berger, R.,Tanzi, R. E., Halperin, J. J., Herzfeldt, B., Van den Bergh, R., Hung, W.-Y.,Bird, T., Deng, G., Mulder, D. W., Smyth, C., Laing, N. G., Soriano, E.,Pericak-Vance, M. A., Haines, J., Rouleau, G. A., Gusella, J. S., Horvitz,H. R., and Brown, R. H., Jr. (1993) Nature 362, 59–62

2. Andersen, P. M., Morita, M., and Brown, R. H., Jr. (2000) in AmyotrophicLateral Sclerosis (Brown, R. H., Jr., Meininger, V., and Swash, M., eds) pp.223–250, Martin Dunitz Ltd., London

3. Cleveland, D. W., and Rothstein, J. D. (2001) Nat. Rev. Neurosci. 2,806–819

4. Hart, P. J. (2006) Curr. Opin. Chem. Biol. 10, 131–1385. Borchelt, D. R., Lee, M. K., Slunt, H. H., Guarnieri, M., Xu, Z.-S., Wong,

P. C., Brown, R. H., Jr., Price, D. L., Sisodia, S. S., and Cleveland, D. W.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8292–8296

6. Borchelt, D. R., Guarnieri, M., Wong, P. C., Lee, M. K., Slunt, H. S., Xu,Z.-S., Sisodia, S. S., Price, D. L., and Cleveland, D. W. (1995) J. Biol. Chem.270, 3234–3238

7. Gurney, M. E., Pu, H., Chiu, A. Y., Dal Canto, M. C., Polchow, C. Y.,Alexander, D. D., Caliendo, J., Hentati, A., Kwon, Y. W., Deng, H.-X.,Chen, W., Zhai, P., Sufit, R. L., and Siddique, T. (1994) Science 264,1772–1775

8. Wong, P. C., Pardo, C. A., Borchelt, D. R., Lee, M. K., Copeland, N. G.,Jenkins, N. A., Sisodia, S. S., Cleveland, D. W., and Price, D. L. (1995)Neuron 14, 1105–1116

9. Reaume, A. G., Elliott, J. L., Hoffman, E. K., Kowall, N. W., Ferrante, R. J.,Siwek, D. F., Wilcox, H. M., Flood, D. G., Beal, M. F., Brown, R. H., Jr.,Scott, R. W., and Snider, W. D. (1996) Nat. Genet. 13, 43–47

10. Pardo, C. A., Xu, Z., Borchelt, D. R., Price, D. L., Sisodia, S. S., and Cleve-land, D. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 954–958

11. Hartz, J. W., and Deutsch, H. F. (1972) J. Biol. Chem. 247, 7043–705012. Fridovich, I. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 35–9713. Parge, H. E., Hallewell, R. A., and Tainer, J. A. (1992) Proc. Natl. Acad. Sci.

U. S. A. 89, 6109–611314. Valentine, J. S., and Hart, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,

3617–362215. Ratovitski, T., Corson, L. B., Strain, J.,Wong, P., Cleveland, D.W., Culotta,

V. C., and Borchelt, D. R. (1999) Hum. Mol. Genet. 8, 1451–146016. Wang, J., Xu, G., Gonzales, V., Coonfield, M., Fromholt, D., Copeland,

N. G., Jenkins, N. A., and Borchelt, D. R. (2002) Neurobiol. Dis. 10,128–138

17. Wang, J., Slunt, H., Gonzales, V., Fromholt, D., Coonfield, M., Copeland,N. G., Jenkins, N. A., and Borchelt, D. R. (2003) Hum. Mol. Genet. 12,2753–2764

18. Elam, J. S., Taylor, A. B., Strange, R., Antonyuk, S., Doucette, P. A., Ro-driguez, J. A., Hasnain, S. S., Hayward, L. J., Valentine, J. S., Yeates, T. O.,and Hart, P. J. (2003) Nat. Struct. Biol. 10, 461–467

19. Antonyuk, S., Elam, J. S., Hough, M. A., Strange, R. W., Doucette, P. A.,Rodriguez, J. A., Hayward, L. J., Valentine, J. S., Hart, P. J., and Hasnain,S. S. (2005) Protein Sci. 14, 1201–1213

20. Liochev, S. I., Chen, L. L., Hallewell, R. A., and Fridovich, I. (1997) Arch.Biochem. Biophys. 346, 263–268

21. Bartnikas, T. B., and Gitlin, J. D. (2003) J. Biol. Chem. 278, 33602–3360822. Laemmli, U. K. (1970) Nature (Lond.) 227, 680–68523. Rumfeldt, J. A., Stathopulos, P. B., Chakrabarrty, A., Lepock, J. R., and

Meiering, E. M. (2006) J. Mol. Biol. 355, 106–12324. Ray, S. S., Nowak, R. J., Strokovich, K., Brown, R. H., Jr., Walz, T., and

Lansbury, P. T., Jr. (2004) Biochemistry 43, 4899–490525. Ripps, M. E., Huntley, G.W., Hof, P. R., Morrison, J. H., and Gordon, J.W.

(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 689–69326. Bruijn, L. I., Becher, M. W., Lee, M. K., Anderson, K. L., Jenkins, N. A.,

Copeland, N. G., Sisodia, S. S., Rothstein, J. D., Borchelt, D. R., Price, D. L.,and Cleveland, D. W. (1997) Neuron 18, 327–338

27. Deng, H. X., Shi, Y., Furukawa, Y., Zhai, H., Fu, R., Liu, E., gorrie, G. H.,Khan, M. S., Hung, W. Y., Bigio, E. H., Lukas, T., Dal Canto, M. C.,

Role of Copper in SOD1 Dimerization and Neurotoxicity

JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 351

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

O’Halloran, T. V., and Siddique, T. (2006) Proc. Natl. Acad. Sci. U. S. A.103, 7142–7147

28. Doucette, P. A., Whitson, L. J., Cao, X., Schirf, V., Demeler, B., Valentine,J. S., Hansen, J. C., and Hart, P. J. (2004) J. Biol. Chem. 279, 54558–54566

29. Pflugrath, J. W. (1999) Acta Crystallogr. D. Biol. Crystallogr. 55,1718–1725

30. Vagin, A., and Teplyakov, A. (2000) Acta Crystallogr. D. Biol. Crystallogr.56, 1622–1624

31. Hart, P. J., Liu, H., Pellegrini,M., Nersissian, A.M., Gralla, E. B., Valentine,J. S., and Eisenberg, D. (1998) Protein Sci. 7, 545–555

32. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P.,Grosse-Kunstleve, R.W., Jiang, J. S., Kuszewski, J., Nilges,M., Pannu,N. S.,Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) ActaCrystallogr. D. Biol. Crystallogr. 54, 905–921

33. Sheldrick, G. M., and Schneider, T. R. (1997) Methods Enzymol. 277,319–343

34. Emsley, P., andCowtan, K. (2004)ActaCrystallogr. D. Biol. Crystallogr. 60,2126–2132

35. Malinowski, D. P., and Fridovich, I. (1979) Biochemistry 18, 237–24436. Vassall, K. A., Stathopulos, P. B., Rumfeldt, J. A., Lepock, J. R., and Meier-

ing, E. M. (2006) Biochemistry 45, 7366–737937. Beauchamp, C., and Fridovich, I. (1971) Anal. Biochem. 44, 276–28738. Hayward, L. J., Rodriguez, J. A., Kim, J. W., Tiwari, A., Goto, J. J., Cabelli,

D. E., Valentine, J. S., and Brown, R. H., Jr. (2002) J. Biol. Chem. 277,15923–15931

39. Furukawa, Y., Torres, A. S., and O’Halloran, T. V. (2004) EMBO J. 23,2872–2881

40. Brown, N.M., Torres, A. S., Doan, P. E., andO’Halloran, T. V. (2004) Proc.Natl. Acad. Sci. U. S. A. 101, 5518–5523

41. Rodriguez, J. A., Shaw, B. F., Durazo, A., Sohn, S. H., Doucette, P. A.,Nersissian, A. M., Faull, K. F., Eggers, D. K., Tiwari, A., Hayward, L. J., andValentine, J. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10516–10521

42. Carroll, M. C., Girouard, J. B., Ulloa, J. L., Subramaniam, J. R.,Wong, P. C.,Valentine, J. S., andCulotta, V. C. (2004) Proc. Natl. Acad. Sci. U. S. A. 101,5964–5969

43. Tainer, J. A., Getzoff, E. D., Beem, K.M., Richardson, J. S., and Richardson,D. C. (1982) J. Mol. Biol. 160, 181–217

44. Aoki, M., Ogasawara, M., Matsubara, Y., Narisawa, K., Nakamura, S.,Itoyama, Y., and Abe, K. (1993) Nat. Genet. 5, 323–324

45. Aoki, M., Ogasawara, M., Matsubara, Y., Narisawa, K., Nakamura, S.,Itoyama, Y., and Abe, K. (1994) J. Neurol. Sci. 126, 77–83

46. Jonsson, P. A., Ernhill, K., Andersen, P. M., Bergemalm, D., Brannstrom,T., Gredal, O., Nilsson, P., and Marklund, S. L. (2004) Brain 127, 73–88

47. Wang, J., Xu, G., Li, H., Gonzales, V., Fromholt, D., Karch, C., Copeland,N. G., Jenkins, N. A., and Borchelt, D. R. (2005) Hum. Mol. Genet. 14,2335–2347

48. Bergemalm, D., Jonsson, P. A., Graffmo, K. S., Andersen, P. M.,Brannstrom, T., Rehnmark, A., andMarklund, S. L. (2006) J. Neurosci. 26,4147–4154

Role of Copper in SOD1 Dimerization and Neurotoxicity

352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 1 • JANUARY 5, 2007

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from

Hart and David R. BorcheltSlunt, Xiaohang Cao, Jonathan Gitlin, P. JohnRodriguez, Jonathan P. Scheurmann, Hilda H. Jiou Wang, Amy Caruano-Yzermans, Angela  Copper and Compromise Dimer StabilityDismutase 1 Diminish the Binding ofLigand Histidine Residues of Superoxide Disease-associated Mutations at CopperProtein Structure and Folding:

doi: 10.1074/jbc.M604503200 originally published online November 8, 20062007, 282:345-352.J. Biol. Chem. 

  10.1074/jbc.M604503200Access the most updated version of this article at doi:

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/282/1/345.full.html#ref-list-1

This article cites 46 references, 21 of which can be accessed free at

by guest on May 15, 2015

http://ww

w.jbc.org/

Dow

nloaded from