Disease-associated Mutations at Copper Ligand Histidine Residues of Superoxide Dismutase 1 Diminish the Binding of Copper and Compromise Dimer Stability

Abstract

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with -ONOO and H2O2 in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind 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 a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry. A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with -ONOO and H2O2 in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind 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 a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry. Amyotrophic lateral sclerosis (ALS), 3The abbreviations used are: ALS, amyotrophic lateral sclerosis; FALS, familial ALS; SOD1, Cu/Zn-superoxide dismutase 1; CHO, Chinese hamster ovary; Quad, quadruple mutant H46R/H48Q/H63G/H120G. which is characterized by progressive muscle weakness and motor neuron loss, presents as both sporadic and familial (FALS) illness. A subset of FALS cases is caused by missense mutations in the superoxide scavenging enzyme, Cu/Zn-superoxide dismutase 1 (SOD1) (1Rosen 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. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J.J. Herzfeldt B. Van den B ergh 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. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar, 2Andersen P.M. Morita M. Brown Jr., R.H. Brown Jr., R.H. Meininger V. Swash M. Amyotrophic Lateral Sclerosis. Martin Dunitz Ltd., London2000: 223-250Google Scholar). To date, over 100 different point mutations, and >5 early termination mutations have been linked to FALS (www.alsod.org) (for reviews see Refs. 2Andersen P.M. Morita M. Brown Jr., R.H. Brown Jr., R.H. Meininger V. Swash M. Amyotrophic Lateral Sclerosis. Martin Dunitz Ltd., London2000: 223-250Google Scholar, 3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1188) Google Scholar, 4Hart P.J. Curr. Opin. Chem. Biol. 2006; 10: 131-138Crossref PubMed Scopus (92) Google Scholar). Early studies of FALS-SOD1 enzymes demonstrated that some mutants retain high levels of activity and relatively long half lives (5Borchelt D.R. Lee M.K. Slunt H.H. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. 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Each subunit of the mature homodimeric SOD1 enzyme binds one atom of copper and one atom of zinc and contains a single oxidized disulfide bond between Cys-57 and Cys-146 (11Hartz J.W. Deutsch H.F. J. Biol. Chem. 1972; 247: 7043-7050Abstract Full Text PDF PubMed Google Scholar, 12Fridovich I. Adv. Enzymol. Relat. Areas Mol. Biol. 1974; 41: 35-97PubMed Google Scholar, 13Parge H.E. Hallewell R.A. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6109-6113Crossref PubMed Scopus (285) Google Scholar). Because copper can participate in many types of potentially deleterious reactions, the of the copper of SOD1 in the with mutant been (for see Refs. 4Hart P.J. Curr. Opin. Chem. Biol. 2006; 10: 131-138Crossref PubMed Scopus (92) Google and J.S. P.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). of or are also to be but this not yet been demonstrated To this in we copper binding of SOD1 H46R, H48Q, and using a direct radioactive copper incorporation In transfected we show that of these variants high for copper. We also single crystal x-ray to the copper binding site of expressed in yeast and by This that the mutant does not bind copper in of the of the We also that of copper ligands with to stable using native gel electrophoresis and a yeast two-hybrid We these as evidence that the of ligands in SOD1 the stable binding of and that the of binding the of these mutants to mature into stable dimeric We that of or proteins found in of enzyme, that the incorporation of copper in these mutants also in of SOD1 the mutations, H46R, H48Q, and into the Each of these been and used in studies (5Borchelt D.R. Lee M.K. Slunt H.H. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. 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Borchelt D.R. 10: PubMed Scopus Google Scholar, J. Slunt H. V. D. M. Copeland N.G. Jenkins N.A. Borchelt D.R. Mol. Genet. PubMed Scopus Google Scholar). Although this mutant superoxide activity J. Xu G. V. M. D. Copeland N.G. Jenkins N.A. Borchelt D.R. 10: PubMed Scopus Google Scholar), this mutant stably binds copper was not we of evidence that that this does not stably bind copper. the crystal of from yeast of copper in the copper or zinc and of the of in the homodimeric enzyme V. B. J.S. P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the from yeast of copper of to cells transfected with for demonstrate or with this as by of Together, these data establish that does not a high for copper. Moreover, we our to SOD1 variants mutations at We find that single mutations at or H48Q, expressed in also diminish the binding of radioactive copper. the mutant also evidence of copper We also find by SOD1 variants with mutations at In native SOD1 variants expressed in cells that mutations at ligands to that of the In the of mutant SOD1 in from mice both the and mutants at a We that and have to stably bind which to also the formation of the normal dimeric enzyme. and of of copper in the site of the mutant is with is in different crystal of SOD1 that in the data which contains a of SOD1 S. J.S. M.A. R.W. J.A. J.S. P.J. S.S. Sci. 14: PubMed Scopus Google Scholar, Chen Hallewell R.A. I. PubMed Scopus Google Scholar). In the of in these a bond to the active site bond the of the or of in or a of S. J.S. M.A. R.W. J.A. J.S. P.J. S.S. Sci. 14: PubMed Scopus Google Scholar, Chen Hallewell R.A. I. PubMed Scopus Google Scholar). these data that the the binding of copper in the copper In the SOD1 been to bind copper at the copper site expressed in the of high levels of copper J.A. J.W. A. Goto J.J. J.S. Brown Jr., R.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or in Chen Hallewell R.A. I. PubMed Scopus Google Scholar). of expressed in cells in with copper and zinc to suggested that the copper is in a that from the found in one that is J.A. J.W. A. Goto J.J. J.S. Brown Jr., R.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of copper ion bound to yeast SOD1 with of copper and zinc was in the x-ray crystal to high In this the copper was found to be with a and the of and in the and as a of the studies evidence that SOD1 is of binding in the copper site in a studies are in with the data in that SOD1 proteins with the to show binding of copper. In to the studies that used we find that the expressed in does not bind copper with and used in at that do not the binding of copper to SOD1 J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although we the that the bound copper from the the of in these was to that of the mutants which are to and In yeast and SOD1, the of the disulfide bond between Cys-57 and Cys-146 the subunit the of enzyme. This of the disulfide bond to be the of copper J. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). enzyme that oxidized disulfide and zinc bound in the zinc the of copper is not to homodimeric enzyme V. B. J.S. P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To produce of both the and the of the normal disulfide bond are V. B. J.S. P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for SOD1, is with both of and of the However, for the mutants we we do not also requires of the disulfide of the data be that the mutants bind copper This with the of the to that for normal dimeric J. PubMed Scopus Google Scholar). of SOD1 gel electrophoresis been used to dimeric and of SOD1, with enzyme dimeric enzyme J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. PubMed Scopus Google Scholar). A in using native is that some FALS mutations can the of dimeric SOD1 (5Borchelt D.R. Lee M.K. Slunt H.H. Guarnieri M. Xu 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: 8292-8296Crossref PubMed Scopus (533) Google Scholar). dimers and (5Borchelt D.R. Lee M.K. Slunt H.H. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. 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Biol. 2006; PubMed Scopus Google Scholar, J.A. 2006; PubMed Scopus Google have established that the and mutants can stable We also that the of from was with dimeric enzyme variant not with in the two-hybrid data with that the variant at S.S. R.J. Brown Jr., R.H. T. Jr., PubMed Scopus Google Scholar). Moreover, studies have demonstrated that to bind copper and the the is J.A. A. A. J.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the data and in the yeast two-hybrid assay are with data these mutants show in the two-hybrid assay, data that are by the of these mutants in native in the high used in some of the dimers the mature enzyme However, at the in or transgenic our data that the of the is We these data as that the variants we have mutations at to a with the formation of stable In SOD1 copper via with the yeast copper for SOD1 or from M.C. J.L. Wong P.C. J.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and we that the proteins used in the yeast two-hybrid copper. we found that the levels of in the yeast used in our assay to of the proteins bound copper active in assay data now We be that the binding of copper by the or proteins is for the to with proteins or that the of copper binding by the mutants is for the to for these proteins to with Because the are not to be in at the H.E. Hallewell R.A. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6109-6113Crossref PubMed Scopus (285) Google Scholar, J.A. J.S. J. Mol. Biol. PubMed Scopus Google Scholar), we that binding of copper by the proteins is one in the and the to of to In we demonstrate that mutations at for the binding of H46R, the of these enzymes to stably bind which to dimeric is of that studies have the in with a of the M. M. S. Nat. Genet. 1993; PubMed Scopus Google Scholar, M. M. S. J. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar), which be as with the that a in this mutant bind copper and be However, in transgenic the of for variants to is to that of variants that stably bind copper J. Xu G. V. M. D. Copeland N.G. Jenkins N.A. Borchelt D.R. 10: PubMed Scopus Google Scholar, J. Slunt H. V. D. M. Copeland N.G. Jenkins N.A. Borchelt D.R. Mol. Genet. PubMed Scopus Google Scholar). To this mutants that bind copper have to to mutants that bind copper and are active in toxic chemistry. in our is that to a in with mechanisms the the that mice mutant of SOD1, including the mutants and of mutant as J. Slunt H. V. D. M. Copeland N.G. Jenkins N.A. Borchelt D.R. Mol. Genet. PubMed Scopus Google Scholar, P.M. D. T. P. PubMed Scopus Google Scholar, J. Xu G. H. V. D. C. Copeland N.G. Jenkins N.A. Borchelt D.R. Mol. Genet. 14: PubMed Scopus Google Scholar, D. P.M. T. A. J. Neurosci. 2006; PubMed Scopus Google Scholar), we that of the mutant is to be one of the mechanisms of