Advertisement

Familial Amyotrophic Lateral Sclerosis-associated Mutations Decrease the Thermal Stability of Distinctly Metallated Species of Human Copper/Zinc Superoxide Dismutase*

Open AccessPublished:February 19, 2002DOI:https://doi.org/10.1074/jbc.M112088200
      We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of “WT-like” FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Δ) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by ∼1–6 °C compared with the corresponding WT SOD1 species. Most of the “metal binding region” FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at ∼4–12 °C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.
      SOD1
      copper/zinc superoxide dismutase
      WT
      wild type
      ALS
      amyotrophic lateral sclerosis
      FALS
      familial ALS
      DSC
      differential scanning calorimetry
      O2·¯
      superoxide
      Copper/zinc superoxide dismutase (SOD1)1 catalyzes the disproportionation of two molecules of superoxide anion (O2·¯) into O2 and H2O2 (
      • McCord J.M.
      • Fridovich I.
      ,
      • Bertini I.
      • Mangani S.
      • Viezzoli M.S.
      ) in all eukaryotic cells. Many specific, highly conserved structural interactions confer upon SOD1 a remarkable thermal stability (
      • Forman H.J.
      • Fridovich I.
      ,
      • Lepock J.R.
      • Arnold L.D.
      • Torrie B.H.
      • Andrews B.
      • Kruuv J.
      ,
      • Roe J.A.
      • Butler A.
      • Scholler D.M.
      • Valentine J.S.
      • Marky L.
      • Breslauer K.J.
      ,
      • Battistoni A.
      • Folcarelli S.
      • Cervoni L.
      • Polizio F.
      • Desideri A.
      • Giartosio A.
      • Rotilio G.
      ) and resistance to chemical denaturation (
      • Malinowski D.P.
      • Fridovich I.
      ,
      • Mach H.
      • Dong Z.
      • Middaugh C.R.
      • Lewis R.V.
      ,
      • Mei G.
      • Rosato N.
      • Silva Jr., N.
      • Rusch R.
      • Gratton E.
      • Savini I.
      • Finazzi-Agro A.
      ).
      Each subunit of homodimeric SOD1 is built upon a flattened β-barrel motif with additional loop regions that contribute to metal ion binding and formation of the active site (
      • Richardson J.
      • Thomas K.A.
      • Rubin B.H.
      • Richardson D.C.
      ). One catalytic copper ion and one buried zinc ion per subunit are bound at the active site on the external surface of the β-barrel. Occupancy of the metal ion binding sites confers greater thermal stabilization to the bovine SOD1 apoenzyme (
      • Forman H.J.
      • Fridovich I.
      ,
      • Lepock J.R.
      • Arnold L.D.
      • Torrie B.H.
      • Andrews B.
      • Kruuv J.
      ). The copper and zinc ions are linked directly via the imidazolate side chain of the shared His-63 residue
      SOD1 residues are numbered according to those in the human enzyme.
      2SOD1 residues are numbered according to those in the human enzyme.
      and indirectly via extended interactions between their respective ligands. SOD1 dimerization is stabilized by optimized hydrophobic interactions at the contact interface between complementary patches on each subunit (
      • Richardson J.
      • Thomas K.A.
      • Rubin B.H.
      • Richardson D.C.
      ,
      • Tainer J.A.
      • Getzoff E.D.
      • Beem K.M.
      • Richardson J.S.
      • Richardson D.C.
      ,
      • Tainer J.A.
      • Getzoff E.D.
      • Richardson J.S.
      • Richardson D.C.
      ). A conserved intrasubunit disulfide bond involving Cys-57 also stabilizes the enzyme by anchoring a loop that forms part of the dimer interface to the β-barrel at Cys-146.
      A subset of SOD1 mutations in familial amyotrophic lateral sclerosis (FALS) have been proposed to destabilize the β-barrel or disrupt dimerization of SOD1 monomers (
      • Deng H.X.
      • Hentati A.
      • Tainer J.A.
      • Zafar I.
      • Cayabyab A.
      • Hung W.Y.
      • Hu E.D.
      • Getzoff P.
      • Herzfeld B.
      • Roos R.P.
      • Warner C.
      • Deng G.
      • Soriano E.
      • Smyth C.
      • Parge H.E.
      • Ahmed A.
      • Roses A.D.
      • Hallewell R.A.
      • Pericak-Vance M.A.
      • Siddique T.
      ,
      • Cleveland D.W.
      • Rothstein J.D.
      ). A crystal structure obtained for the G37R SOD1 mutant shows minimal perturbation of the averaged backbone conformation but exhibits unusually high atomic displacement parameters, suggestive of increased molecular flexibility in some regions of the molecule (
      • Hart P.J.
      • Liu H.
      • Pellegrini M.
      • Nersissian A.M.
      • Gralla E.B.
      • Valentine J.S.
      • Eisenberg D.
      ). Consistent with this, some mutant SOD1s exhibit accelerated turnover in vivo (
      • Borchelt D.R.
      • Lee M.K.
      • Slunt H.S.
      • Xu M.
      • Guarnieri Z.S.
      • Wong P.C.
      • Brown Jr., R.H.
      • Price D.L.
      • Sisodia S.S.
      • Cleveland D.W.
      ,
      • Ratovitski T.
      • Corson L.B.
      • Strain J.
      • Wong P.
      • Cleveland D.W.
      • Culotta V.C.
      • Borchelt D.R.
      ) or increased susceptibility to proteolytic digestion (
      • Ratovitski T.
      • Corson L.B.
      • Strain J.
      • Wong P.
      • Cleveland D.W.
      • Culotta V.C.
      • Borchelt D.R.
      ) compared with the wild type (WT) enzyme. However, a quantitative analysis of FALS mutant SOD1 structural stability and its relation to metal occupancy has not been reported.
      In the present study, we investigated the effects of 14 ALS-associated mutations on the thermodynamic stability of biologically metallated human SOD1s. Using differential scanning calorimetry (DSC), we measured the melting temperatures (Tm) of WT SOD1 and mutant variants hypothesized to perturb the active site region, the dimer interface, one pole of the β-barrel, and associated loops. We distinguished the melting properties of partially metallated SOD1 species and showed that mutant SOD1 proteins were destabilized relative to the corresponding WT species.

      DISCUSSION

      We measured the thermal unfolding behavior of partially metallated species of WT and 14 mutant SOD1s associated with familial ALS (TableI). Copper and zinc ions were more likely to be bound to their correct sites in these biologically metallated proteins compared with SOD1 proteins remetallated in vitro (
      • Hayward L.J.
      • Rodriguez J.A.
      • Kim J.W.
      • Tiwari A.
      • Goto J.J.
      • Cabelli D.E.
      • Valentine J.S.
      • Brown Jr., R.H.
      ,
      • Goto J.J.
      • Zhu H.
      • Sanchez R.J.
      • Nersissian A.
      • Gralla E.B.
      • Valentine J.S.
      • Cabelli D.E.
      ). Our initial experiments using DSC correlated increased metal contents with the unfolding of more stable SOD1 species (Fig. 2). For the WT and A4V mutants, we also identified the unfolding of copper-containing species at Tm3, whereas transitions atTm1 andTm2 most likely resulted from melting of SOD1 subunits lacking copper (Fig. 3).
      Eight of the FALS-associated mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Δ) shared copper coordination and specific activities comparable with WT SOD1 (
      • Hayward L.J.
      • Rodriguez J.A.
      • Kim J.W.
      • Tiwari A.
      • Goto J.J.
      • Cabelli D.E.
      • Valentine J.S.
      • Brown Jr., R.H.
      ). For these “WT-like” mutants, Tm2 was consistently ∼14–16 °C higher than Tm1, whileTm3 was consistently ∼21–24 °C higher than Tm1 (Table I). These results indicate that each mutant in this group preserved the structural interactions important for metal-induced stabilization. However, these WT-like mutants all exhibited reduced thermal stabilities when compared with corresponding WT SOD1 species (Table I).
      Heating of four mutant SOD1s (G93A, G41S, G72S, and D76Y) produced an additional endotherm near 90 °C (Tm4). The appearance of this transition correlated with the mutant proteins containing the highest amounts of copper (Table I). Fully metallated bovine SOD1 unfolds at a similar temperature (∼92 °C) when heated at a comparable rate and concentration (
      • Roe J.A.
      • Butler A.
      • Scholler D.M.
      • Valentine J.S.
      • Marky L.
      • Breslauer K.J.
      ). We hypothesize that the transition atTm4 in our study arose from a subpopulation of SOD1s containing two Cu(II) and two Zn(II) ions per dimer, whereas Tm3 reflected the unfolding of partially metallated species that contained copper.
      A previous DSC analysis by Lepock et al. (
      • Lepock J.R.
      • Frey H.E.
      • Hallewell R.A.
      ) of recombinant WT human SOD1 expressed in yeast produced major endotherms at 74.9 °C (component 1) and 83.6 °C (component 2). These transitions were assumed to arise from fully metallated species of SOD1 containing oxidized and reduced copper, respectively, but the total metal contents and the oxidation states of copper were not reported. Our results are consistent with those of Lepock et al. (
      • Lepock J.R.
      • Frey H.E.
      • Hallewell R.A.
      ) if components 1 and 2 in their study correspond to the transitions at Tm2 (species lacking copper but probably stabilized by zinc) andTm3 (partially metallated species containing copper) observed for WT SOD1 in the present work. This conclusion is also supported by the finding that human SOD1 proteins expressed in yeast may be undermetallated (
      • Goto J.J.
      • Gralla E.B.
      • Valentine J.S.
      • Cabelli D.E.
      ).
      The Tm of a SOD1 subunit within a dimeric species probably depends on both its metal occupancy and its interactions with the partner subunit. In addition, the heating procedure during DSC may favor the rearrangement or consolidation of SOD1 species into more stable dimers (
      • Brandts J.F.
      • Lin L.N.
      ). For example, denaturation or dissociation of a less stable subunit in a partially metallated dimer during slow heating would be expected also to decrease the stability of the partner subunit upon loss of strong intersubunit hydrophobic interactions. If monomerization of the partner occurs during heating, exposure of its hydrophobic dimer interface to the solvent would be thermodynamically unfavorable. The partner subunit may retain its full stability by reassociation with a similarly stable subunit to produce a new population of more completely metallated dimers.
      Why did we observe a maximum of four distinct unfolding transitions for the biologically metallated SOD1 enzymes? If metal ions are incorporated into their proper sites, such that four potential metallation states exist for each SOD1 subunit (E-E, Cu-E, E-Zn, or Cu-Zn, where “E” refers to an empty metal binding site), then a total of 10 distinctly metallated dimeric SOD1 species or four monomeric species could be present in our samples. It is possible that many of these species exist but that the melting transitions within specific subsets overlap sufficiently to become indistinguishable by DSC. Alternatively, only a fraction of these possible species may occurin vivo, since metal ion incorporation into SOD1 may be a cooperative process (
      • Roe J.A.
      • Butler A.
      • Scholler D.M.
      • Valentine J.S.
      • Marky L.
      • Breslauer K.J.
      ). Prior stabilization by zinc binding, for example, might be important for biological incorporation of copper into SOD1.
      Many of the mechanisms that have been proposed to explain FALS mutant SOD1 toxicity (
      • Cleveland D.W.
      • Rothstein J.D.
      ) presume an altered conformation of mutant compared with WT SOD1. Decreased mutant SOD1 stability may help to explain how >90 mutations scattered over more than one-third of the residues in this enzyme all cause a similar phenotype. The extreme thermochemical stability of fully metallated normal SOD1 implies that selection has occurred against less stable forms of the enzyme.
      We have shown here that some FALS-associated SOD1 mutants are less stable than corresponding WT forms despite binding similar amounts of metal ions. We hypothesize that the observed thermal destabilization of the mutant enzymes in vitro may parallel a susceptibility to specific biochemical denaturing stresses in vivo. Although mutants such as D90A or G93A exhibited only a mild reduction in global stability as measured by DSC, it is possible that a more localized instability may be pertinent to mutant SOD1 toxicity. Consistent with this view, D90A SOD1 exhibits an accelerated loss of dismutase activity compared with WT SOD1 upon exposure to either high temperature (73 °C, ∼3-fold acceleration) or denaturant (3.5 mguanidinium chloride, ∼6-fold acceleration) (
      • Marklund S.L.
      • Andersen P.M.
      • Forsgren L.
      • Nilsson P.
      • Ohlsson P.I.
      • Wikander G.
      • Oberg A.
      ). While loss of dismutase activity itself is unlikely to be the toxic property of D90A SOD1, it may reflect a more ominous tendency of the enzyme to unfold locally.
      For other SOD1 mutants, a decreased metal content greatly increases the fraction of destabilized protein. Six of the mutant proteins investigated here had substitutions that either perturb a copper ligand (H46R and H48Q) or may disrupt other important motifs in the metal binding region (G85R, D124V, D125H, and S134N). It is possible that metal loss from mutant SOD1 in the presence of other cellular denaturing influences may contribute to an increased burden of misfolded proteins at physiological temperatures. Indeed, recent analyses of SOD1 transgenic mice (
      • Watanabe M.
      • Dykes-Hoberg M.
      • Cizewski Culotta V.
      • Price D.L.
      • Wong P.C.
      • Rothstein J.D.
      ) or rats (
      • Nagai M.
      • Aoki M.
      • Miyoshi I.
      • Kato M.
      • Pasinelli P.
      • Kasai N.
      • Brown Jr., R.H.
      • Itoyama Y.
      ) indicate that those models expressing the metal-binding region mutants (H46R or G85R) exhibit frequent inclusion bodies recognized by antibodies to SOD1 or ubiquitin, while those models expressing the β-barrel pole mutants (G37R or G93A) exhibit fewer inclusions but more prominent vacuolar changes.
      Our investigations of thermal stability for a large group of SOD1 variants associated with familial ALS have led to three significant observations: 1) distinct species within heterogeneous mixtures of partially metallated SOD1 can be effectively compared using DSC; 2) many mutants retain the ability to be stabilized upon metal ion binding or to be destabilized upon metal loss; and 3) all of the mutant proteins exhibit some degree of reduced stability compared with WT SOD1. The contribution of decreased SOD1 conformational stability to the pathogenesis of familial ALS remains to be explored in greater cellular and molecular detail.

      REFERENCES

        • McCord J.M.
        • Fridovich I.
        J. Biol. Chem. 1969; 244: 6049-6055
        • Bertini I.
        • Mangani S.
        • Viezzoli M.S.
        Adv. Inorg. Chem. 1998; 45: 127-250
        • Forman H.J.
        • Fridovich I.
        J. Biol. Chem. 1973; 248: 2645-2649
        • Lepock J.R.
        • Arnold L.D.
        • Torrie B.H.
        • Andrews B.
        • Kruuv J.
        Arch. Biochem. Biophys. 1985; 241: 243-251
        • Roe J.A.
        • Butler A.
        • Scholler D.M.
        • Valentine J.S.
        • Marky L.
        • Breslauer K.J.
        Biochemistry. 1988; 27: 950-958
        • Battistoni A.
        • Folcarelli S.
        • Cervoni L.
        • Polizio F.
        • Desideri A.
        • Giartosio A.
        • Rotilio G.
        J. Biol. Chem. 1998; 273: 5655-5661
        • Malinowski D.P.
        • Fridovich I.
        Biochemistry. 1979; 18: 5055-5060
        • Mach H.
        • Dong Z.
        • Middaugh C.R.
        • Lewis R.V.
        Arch. Biochem. Biophys. 1991; 287: 41-47
        • Mei G.
        • Rosato N.
        • Silva Jr., N.
        • Rusch R.
        • Gratton E.
        • Savini I.
        • Finazzi-Agro A.
        Biochemistry. 1992; 31: 7224-7230
        • Richardson J.
        • Thomas K.A.
        • Rubin B.H.
        • Richardson D.C.
        Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1349-1353
        • Tainer J.A.
        • Getzoff E.D.
        • Beem K.M.
        • Richardson J.S.
        • Richardson D.C.
        J. Mol. Biol. 1982; 160: 181-217
        • Tainer J.A.
        • Getzoff E.D.
        • Richardson J.S.
        • Richardson D.C.
        Nature. 1983; 306: 284-287
        • Deng H.X.
        • Hentati A.
        • Tainer J.A.
        • Zafar I.
        • Cayabyab A.
        • Hung W.Y.
        • Hu E.D.
        • Getzoff P.
        • Herzfeld B.
        • Roos R.P.
        • Warner C.
        • Deng G.
        • Soriano E.
        • Smyth C.
        • Parge H.E.
        • Ahmed A.
        • Roses A.D.
        • Hallewell R.A.
        • Pericak-Vance M.A.
        • Siddique T.
        Science. 1993; 261: 1047-1051
        • Cleveland D.W.
        • Rothstein J.D.
        Nat. Rev. Neurosci. 2001; 2: 806-819
        • Hart P.J.
        • Liu H.
        • Pellegrini M.
        • Nersissian A.M.
        • Gralla E.B.
        • Valentine J.S.
        • Eisenberg D.
        Protein Sci. 1998; 7: 545-555
        • Borchelt D.R.
        • Lee M.K.
        • Slunt H.S.
        • Xu M.
        • Guarnieri Z.S.
        • Wong P.C.
        • Brown Jr., R.H.
        • Price D.L.
        • Sisodia S.S.
        • Cleveland D.W.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296
        • Ratovitski T.
        • Corson L.B.
        • Strain J.
        • Wong P.
        • Cleveland D.W.
        • Culotta V.C.
        • Borchelt D.R.
        Hum. Mol. Genet. 1999; 8: 1451-1460
        • Hayward L.J.
        • Rodriguez J.A.
        • Kim J.W.
        • Tiwari A.
        • Goto J.J.
        • Cabelli D.E.
        • Valentine J.S.
        • Brown Jr., R.H.
        J. Biol. Chem. 2002; 277: 15923-15931
        • Goto J.J.
        • Gralla E.B.
        • Valentine J.S.
        • Cabelli D.E.
        J. Biol. Chem. 1998; 273: 30104-30109
        • Pace C.N.
        • Vajdos F.
        • Fee L.
        • Grimsley G.
        • Gray T.
        Protein Sci. 1995; 4: 2411-2423
        • Edge V.
        • Allewell N.M.
        • Sturtevant J.M.
        Biochemistry. 1985; 24: 5899-5906
        • Lepock J.R.
        • Frey H.E.
        • Hallewell R.A.
        J. Biol. Chem. 1990; 265: 21612-21618
        • Liu H.
        • Zhu H.
        • Eggers D.K.
        • Nersissian A.M.
        • Faull K.F.
        • Ai J.J.
        • Goto J.
        • Sanders-Loehr J.
        • Gralla E.B.
        • Valentine J.S.
        Biochemistry. 2000; 39: 8125-8132
        • Goto J.J.
        • Zhu H.
        • Sanchez R.J.
        • Nersissian A.
        • Gralla E.B.
        • Valentine J.S.
        • Cabelli D.E.
        J. Biol. Chem. 2000; 275: 1007-1014
        • Brandts J.F.
        • Lin L.N.
        Biochemistry. 1990; 29: 6927-6940
        • Marklund S.L.
        • Andersen P.M.
        • Forsgren L.
        • Nilsson P.
        • Ohlsson P.I.
        • Wikander G.
        • Oberg A.
        J. Neurochem. 1997; 69: 675-681
        • Watanabe M.
        • Dykes-Hoberg M.
        • Cizewski Culotta V.
        • Price D.L.
        • Wong P.C.
        • Rothstein J.D.
        Neurobiol. Dis. 2001; 8: 933-941
        • Nagai M.
        • Aoki M.
        • Miyoshi I.
        • Kato M.
        • Pasinelli P.
        • Kasai N.
        • Brown Jr., R.H.
        • Itoyama Y.
        J. Neurosci. 2001; 21: 9246-9254
        • Hosler B.A.
        • Nicholson G.A.
        • Sapp P.C.
        • Chin W.
        • Orrell R.W.
        • de Belleroche J.S.
        • Esteban J.
        • Hayward L.J.
        • McKenna-Yasek D.
        • Yeung L.
        • Cherryson A.K.
        • Dench J.E.
        • Wilton S.D.
        • Laing N.G.
        • Horvitz R.H.
        • Brown Jr., R.H.
        Neuromusc. Disord. 1996; 6: 361-366