The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status
The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis. The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis. Eukaryotic copper,zinc superoxide dismutase (SOD1) 1The abbreviations used are: SOD1, Cu,Zn-superoxide dismutase; hSOD1, human SOD1; ySOD1, yeast SOD1; E,E-hSOD1SH, fully reduced and demetallated hSOD1; E,Zn-hSOD1SH, fully reduced Znloaded hSOD1; E,E-hSOD1SS, oxidized and demetallated hSOD1; E,Zn-hSOD1SS, oxidized and Zn-loaded hSOD1; Q133M2SOD1, human SOD1 with the mutations F50E/G51E/E133Q; CCS, copper chaperone for SOD1; AMS, 4-acetamide-4′maleimidylstilbene-2,2′-disulfonic acid; WT, wild type; HSQC, heteronuclear single quantum coherence; CD, circular dichroism; fALS, familial form of amyotrophic lateral sclerosis.1The abbreviations used are: SOD1, Cu,Zn-superoxide dismutase; hSOD1, human SOD1; ySOD1, yeast SOD1; E,E-hSOD1SH, fully reduced and demetallated hSOD1; E,Zn-hSOD1SH, fully reduced Znloaded hSOD1; E,E-hSOD1SS, oxidized and demetallated hSOD1; E,Zn-hSOD1SS, oxidized and Zn-loaded hSOD1; Q133M2SOD1, human SOD1 with the mutations F50E/G51E/E133Q; CCS, copper chaperone for SOD1; AMS, 4-acetamide-4′maleimidylstilbene-2,2′-disulfonic acid; WT, wild type; HSQC, heteronuclear single quantum coherence; CD, circular dichroism; fALS, familial form of amyotrophic lateral sclerosis. catalyzes the dismutation of superoxide radical to oxygen and hydrogen peroxide and is a 32-kDa homodimeric enzyme found predominantly in the cytosol (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). SOD1 is one of the most thermally stable enzymes known in mesophilic organisms. Dismutase activity declines at 80 °C with a corresponding melting temperature, Tm, above 90 °C (2Roe J.A. Butler A. Scholler D.M. Valentine J.S. Marky L. Breslauer K.J. Biochemistry. 1988; 27: 950-958Crossref PubMed Scopus (109) Google Scholar). The protein is stable in the presence of strong denaturants, and the activity is observed in 4% SDS or 10 m urea (3Forman H.J. Fridovich I. J. Biol. Chem. 1973; 248: 2645-2649Abstract Full Text PDF PubMed Google Scholar). Structural properties of SOD1 that contribute to this extreme thermochemical stability are thought to include an eight-stranded β-barrel motif, hydrophobic interactions associated with dimerization, coordinate covalent bonds, and an intrasubunit disulfide bond between highly conserved pair of cysteines, namely Cys57 and Cys146 in the human form. Whereas the dimerization can contribute to the structural stability through the reduction of its mobility (4Banci L. Bertini I. Cramaro F. Del Conte R. Rosato A. Viezzoli M.S. Biochemistry. 2000; 39: 9108-9118Crossref PubMed Scopus (57) Google Scholar), the roles of the disulfide bond in the SOD1 function and/or structure are only now beginning to emerge. Inspection of the SOD1 structure reveals that the loop containing Cys57 can influence the conformation of the catalytically important residue, Arg143, through a hydrogen-bonding network (5Fisher C.L. Cabelli D.E. Tainer J.A. Hallewell R.A. Getzoff E.D. Proteins. 1994; 19: 24-34Crossref PubMed Scopus (111) Google Scholar). Portions of this loop contribute to the dimer interface (6Bertini I. Mangani S. Viezzoli M.S. Sykes A.G. Advanced Inorganic Chemistry. Academic Press, San Diego1998: 127-250Google Scholar), leading to the possibility that the disulfide bond influences the protein dimerization and thereby the SOD1 quaternary structure. To attain the correctly folded quaternary structure and become enzymatically active, several post-translational modifications need to occur in SOD1 such as the acquisition of copper and zinc ions, formation of the disulfide bond, and dimerization. Whereas the mechanism by which SOD1 acquires Zn(II) is not fully understood, several aspects of the copper insertion by the copper chaperone for SOD1 (CCS) are well established (7O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2000; 275: 25057-25060Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 8Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1358) Google Scholar, 9Rae T.D. Torres A.S. Pufahl R.A. O'Halloran T.V. J. Biol. Chem. 2001; 276: 5166-5176Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Biochemistry. 2000; 39: 14720-14727Crossref PubMed Scopus (89) Google Scholar, 11Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 12Eisses J.F. Stasser J.P. Ralle M. Kaplan J.H. Blackburn N.J. Biochemistry. 2000; 39: 7337-7342Crossref PubMed Scopus (55) Google Scholar). More recently, Furukawa et al. (13Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (291) Google Scholar) have shown that the intrasubunit disulfide bond is correctly introduced in yeast SOD1 by the copper-bound form of yeast CCS. However, given that the cytosol is a strongly reducing environment due to the high GSH/GSSG ratio (100:1–1000:1) (14Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1580) Google Scholar), the disulfide formation is an unfavorable process. Those results suggest that the immature disulfide-reduced hSOD1 is a more important species in the cytosolic environment than previously thought. Field et al. (15Field L.S. Furukawa Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; 278: 28052-28059Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) have also recently shown that uptake of the SOD1 molecule into the intermembrane space of the mitochondria is dependent on the status of the disulfide bond. The reduced form of SOD1 is imported through the mitochondrial outer membrane, but the disulfide-bonded apo-SOD1, the Zn(II)-loaded SOD1, and the holo-form or fully mature form of SOD1 are not readily transferred from the cytosol into the intermembrane space of the mitochondria. The effects of disulfide reduction on the SOD1 structure are therefore relevant to our understanding of the intracellular localization and stability of the SOD1 molecule. In this study we show that, even after removal of both copper and zinc ions from the active and mature form of hSOD1, the dimeric state still persists; however, upon reduction of the disulfide bond, the protein can readily dissociate to the monomer form. Zn(II) addition to the reduced apo-hSOD1 restores the dimeric state, indicating that only the most immature form of hSOD1 before any post-translational modifications favors the monomeric state. These results provide a molecular basis for understanding factors that control the SOD1 monomer-dimer equilibrum in the cytosol and have direct relevance to models for the toxic gain of function mutations in SOD1 that are associated with familial amyotrophic lateral sclerosis (fALS). Sample Preparation—hSOD1 was expressed in the Escherichia coli TOPP1 (Stratagene) or BL21(DE3) strain. The mutations were performed using a QuikChange™ site-directed mutagenesis kit (Stratagene). The 15N-labeled protein in which the non-conserved cysteine residues, Cys6 and Cys111, were mutated to Ser was obtained by growing the cells in the M9 minimal medium with N15-NH4Cl following a reported procedure (16Banci L. Benedetto M. Bertini I. Del Conte R. Piccioli M. Viezzoli M.S. Biochemistry. 1998; 37: 11780-11791Crossref PubMed Scopus (117) Google Scholar), whereas LB medium was used for the non-labeled protein. The cells were grown at 37 °C until A600 = 0.6 and induced with 1.0 mm isopropyl 1-thio-β-d-galactopyranoside for 6 h. The protein was isolated and purified according to previously published protocols (16Banci L. Benedetto M. Bertini I. Del Conte R. Piccioli M. Viezzoli M.S. Biochemistry. 1998; 37: 11780-11791Crossref PubMed Scopus (117) Google Scholar). Fully reduced and demetallated hSOD1 (E,E-hSOD1SH) was prepared by treating the isolated protein with dithiothreitol at 37 °C for 1 h in an anaerobic chamber to reduce the disulfide bond (13Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (291) Google Scholar). The protein solution was then acidified using 0.4% trifluoroacetic acid, and organic solvents (15% CH3CN, 10% CH3OH) were included to remove the metal ions. The protein solution was purified using reverse-phase high pressure liquid chromatography through a 300-Å C18 Jupiter column (Phenomenex) equilibrated with 0.1% trifluoroacetic acid in water. The fractions containing hSOD1 were eluted with a linear gradient of 0.1% trifluoroacetic acid in CH3CN and lyophilized. The metal content of E,E-hSOD1 was checked by inductively coupled plasma atomic emission spectrometry using a Thermo Jarrell Ash Atomscan Model 25 Sequential inductively coupled spectrometer, and zinc and copper ions were <10 nm in the 2 μm protein sample. Zinc reconstitution was obtained as previously described (17Beem K.M. Rich W.E. Rajagopalan K.V. J. Biol. Chem. 1974; 249: 7298-7305Abstract Full Text PDF PubMed Google Scholar). Protein reduction and metallation were carried out under a nitrogen atmosphere in an anaerobic chamber. Thiol-Disulfide Reduction Assay—The thiol-disulfide status of purified hSOD1 was determined by chemical modification with the thiolspecific reagent acid (13Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (291) Google Scholar). results in a in the molecular of hSOD1 as by and of the SOD1 protein that is in 10 of the was with of mm and of 10% The was at 37 °C for an in an anaerobic and then the without any reducing was at °C for 2 the was on of hSOD1 were on with a of 1 mm was used for the and the were as 2 and 2 was obtained as the of The protein was to the of the of the were by the from the were then using or of the secondary structure were using the C. Google Scholar). were on spectrometer, at a of with the was The and were on mm of 15N-labeled and in mm The and and were as previously described L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar). The of was from the ratio with the R. Science. PubMed Scopus Google Scholar). of the were at using the of the were prepared under nitrogen atmosphere in a were into with μm hSOD1 protein was on at The column was with mm and the was 1.0 To the of the 1 mm dithiothreitol was in the above for the of and the using mm was included in the The was obtained by the at The of the column for the of molecular was performed using of coli and as protein reduction of the disulfide in hSOD1 with dithiothreitol by to remove metal ions the fully reduced and demetallated as by modification and inductively coupled plasma atomic emission spectrometry In the and of the four in hSOD1 have mutated to Ser (i.e. to the of the and reported that these residues, Cys6 and Cys111, which are not in disulfide have effects on the SOD1 activity and structure Hallewell R.A. Tainer J.A. S. A. 1992; PubMed Scopus Google Scholar). the disulfide bond is have shown that SOD1 is of and structure L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar). To the structural upon disulfide was used to the secondary structure. Reduction a on the SOD1 in the of the most immature E,E-hSOD1SH, a at indicating that is predominantly of the Biochemistry. 1969; PubMed Scopus Google Scholar). The of a strong at nm in the low content Biochemistry. 1969; PubMed Scopus Google Scholar). We to any in the secondary structure upon the disulfide formation be by using of the monomeric hSOD1 However, both and to that of the form of the The of C. Google Scholar, Biochemistry. 1974; PubMed Scopus Google Scholar) that these forms of hSOD1 have secondary structure content as reported in I. reduction does not alter the secondary structure, that several of the are before any post-translational We also the effects of the disulfide reduction on the tertiary and quaternary structure of hSOD1 by structure content of forms of hSOD1 obtained from the of by the C. Google Scholar) using the least of et al. Biochemistry. 1974; PubMed Scopus Google in a Reduction and Zinc SOD1 of is shown in several are in a of and in the this is remarkably to that of for which the have L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar). This finding that the tertiary structure of also is to that of i.e. the structure is and and are L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar). loop in Zn(II) and also a residue, namely is that zinc removal and disulfide reduction can and the structural mobility of loop reported that loop which an for uptake of the superoxide well upon the zinc L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar, L. Bertini I. F. M. Viezzoli M.S. Protein PubMed Scopus Google Scholar). even after and reduction of the disulfide bond, most of the protein tertiary structure is with the of the of loop However, zinc is to the a dramatic change in the is observed with the state, a in the which that of the the in the between monomeric and dimeric The and chemical shift between the forms of the protein are shown in as the chemical shift and are chemical shift for and between and are due to disulfide reduction and/or dimerization, between and are due to disulfide reduction and/or copper and between and are due to dimerization and/or copper In of the significant chemical shift are in and whereas for the at the and are The disulfide in the proteins and more than the and which are at the dimer of of these at the dimer which are the and well in are with in The for molecular provides into the hSOD1 quaternary structure. The were from the of and which are obtained for and of and E,Zn-hSOD1SH, The is highly to the protein A. on the of the Scholar), and the dimerization is to from the is in than in In the of is to that of monomeric L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar) and L. Bertini I. F. M. Viezzoli M.S. Protein PubMed Scopus Google Scholar), whereas a to that found for dimeric (4Banci L. Bertini I. Cramaro F. Del Conte R. Rosato A. Viezzoli M.S. Biochemistry. 2000; 39: 9108-9118Crossref PubMed Scopus (57) Google Scholar). These results suggest that is monomeric and that Zn(II) addition to the reduced protein can lead to the dimerization. This is have shown that SOD1 dimer is even after H.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). To the effects of the disulfide reduction on the monomer-dimer equilibrium at physiological concentrations of we and and the at at at at at at at at at at at at L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google L. Bertini I. F. M. Viezzoli M.S. Protein PubMed Scopus Google L. Bertini I. Cramaro F. Del Conte R. Rosato A. Viezzoli M.S. Biochemistry. 2000; 39: 9108-9118Crossref PubMed Scopus (57) Google in a of and the between the and The of reduced favors the monomeric state at the of μm in The physiological of SOD1 in the to be μm in yeast by T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1358) Google Scholar) and μm in the cytosol of by H.J. J.D. J. Biol. 1988; PubMed Scopus Google Scholar). is with an of zinc for an at 37 the strongly favors the dimeric state in The for the monomeric state in can be also by using a hSOD1 mutant in which of the are to (i.e. the The protein conformation of reduced hSOD1 can be by this which any of with the the of this mutant favors the monomeric state and to the dimeric state upon the addition of the zinc To the effects of the conserved disulfide bond on monomer-dimer the non-conserved cysteine residues, Cys6 and Cys111, were mutated to in the the of the mutant favors the monomeric state the disulfide bond is reduced and the addition of can the protein These results show that the monomer-dimer equilibrium is not by the non-conserved cysteine In the conserved are oxidized to form the disulfide bond, the form at the corresponding to the dimeric state in This that, in the of any metal ions, the SOD1 is by reduction of the disulfide. The addition of Zn(II) to does not change the and still favors the dimeric state in Zn(II) removal the hSOD1 protein the disulfide is We conclude that the or folded form of the hSOD1 favors the monomer state until the of several post-translational modifications in the The of copper,zinc superoxide dismutase are not by covalent but are to is observed under extreme denaturing such as m m or SDS (3Forman H.J. Fridovich I. J. Biol. Chem. 1973; 248: 2645-2649Abstract Full Text PDF PubMed Google Scholar). is that the protein is monomeric, even without any In we find that the form is monomeric even at the high concentrations used in the we that μm is in the dimeric state, the for the constant is as 2 the still favors the dimeric state, even at the low concentrations used in the on a of the in the of the as the monomer in this leading to an of the of disulfide formation does not change the secondary structure of hSOD1 favors dimerization by at least four orders of magnitude. The show that the disulfide bond a significant but not in the quaternary structure of the human form of Zinc acquisition by the state to have as on the monomer-dimer equilibrium as disulfide i.e. both and the dimeric state, even protein is as low as 10 Zinc shown to reduce the mobility of the loop with in L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar, L. Bertini I. F. M. Viezzoli M.S. Protein PubMed Scopus Google Scholar), which the important acid for zinc i.e. and with in loop in SOD1 is to the interface between the such a of loop upon the of the is also to important roles in the SOD1 dimerization L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Biochemistry. 2003; PubMed Scopus Google Scholar, L. Bertini I. F. M. Viezzoli M.S. Protein PubMed Scopus Google Scholar). The results show that, even the disulfide is Zn(II) is to a conformation of the protein that favors dimer is to that loop is to the the disulfide formation between Cys57 and Whereas a structural of several SOD1 is in reduction of the disulfide bond and loop in the of its a structure of loop may the between the both disulfide formation and Zn(II) can structure to the protein conformation loop which the between the roles of the disulfide bond in the monomer-dimer equilibrium have also reported in the yeast SOD1 and are in with this study on human However, Furukawa et al. (13Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (291) Google Scholar) have found that reduction of the disulfide in leads to conformation that the monomeric state under the conditions. is an in the of the yeast and human The of reduced human protein favors the dimeric state, whereas the state of the yeast protein favors the monomer state. the structural of and is we that this may be to residues, and the intrasubunit disulfide bond that are in the yeast but not in the human protein. Culotta and Valentine J.S. Culotta V.C. S. A. 2004; PubMed Scopus Google Scholar) have recently shown that these a in the which for the yeast and human The of these the the disulfide bond and loop to reduce the between the that the cytosol can provide the strongly reducing of the GSH/GSSG the monomeric form of this disulfide-reduced SOD1 protein to be more relevant than to In the yeast the most immature i.e. is the only one that is from the cytosol into the intermembrane space of mitochondria. of SOD1 is dependent upon its by yeast the intermembrane space of mitochondria (15Field L.S. Furukawa Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; 278: 28052-28059Abstract Full Text Full Text PDF PubMed Scopus (182) Google however, the Zn(II) is or the disulfide bond is introduced in mitochondrial import of the SOD1 protein is (15Field L.S. Furukawa Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; 278: 28052-28059Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This selectivity for SOD1 mitochondrial import be by our The demetallated and disulfide-reduced form a than any is to be to and its through in the mitochondrial outer that metallation and disulfide formation may the SOD1 dimer from the mitochondrial of the SOD1 quaternary structure by disulfide reduction may be relevant to the of fALS, which associated with a of mitochondrial in J.P. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). mutations in the human SOD1 can cause of fALS, which is a and A. Tainer J.A. A. Getzoff E.D. B. et PubMed Scopus Google Scholar, J. A. Science. 1994; PubMed Scopus Google Scholar). The not the SOD1 activity but gain to cause the such as activity or protein J.P. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that the of the mutant which with L. M. S. A. PubMed Scopus Google Scholar). that protein a in formation of leading to protein R. J.P. A. J. Biol. 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