Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Yoshiaki Furukawa,Itsuki Anzai,Shuji Akiyama,Mizue Imai,Fatima Joy C Cruz,Tomohide Saio,Kenichi Nagasawa,Takao Nomura,Koichiro Ishimori

doi:10.1074/jbc.m115.683763

Abstract

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

Highlights

Mutations in Cu,Zn-superoxide dismutase (SOD1)2 are linked to familial forms of amyotrophic lateral sclerosis [1]
Nuclear Magnetic Resonance—15N-Labeled SOD1 proteins were prepared by culturing E. coli SHuffleTM harboring the plasmid for expression of His-tagged SOD1, and the protein expression was induced with 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside in M9 minimal media containing 15NH4Cl, 3 ␮M ZnSO4, and 30 ␮M CuSO4 at 20 °C for 43 h
To avoid oxidative modifications during experiments, Cys-6 and -111 of SOD1 in this study were mutated to Ser (SOD1(57/146)); the presence of a Cys-57–Cys-146 disulfide bond in SOD1(57/146) was confirmed electrophoretically (SOD1(57/146)S-S)

Summary

Experimental Procedures

Preparation of SOD1 Proteins—Escherichia coli SHuffleTM (New England BioLabs) was transformed with a pET15b plasmid containing cDNA of human SOD1 with an N-terminal His tag, and the expression of His-tagged SOD1 proteins was induced by culturing the transformed cells in the presence of 0.1 mM isopropyl ␤-D-1-thiogalactopyranoside at 20 °C for 16 h. Nuclear Magnetic Resonance—15N-Labeled SOD1 proteins were prepared by culturing E. coli SHuffleTM harboring the plasmid for expression of His-tagged SOD1, and the protein expression was induced with 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside in M9 minimal media containing 15NH4Cl, 3 ␮M ZnSO4, and 30 ␮M CuSO4 at 20 °C for 43 h His tag-free demetallated SOD1 proteins were purified as described above. SAXS measurements were performed at 10 °C using a series of samples serially diluted from 6.58 to 2.53 g/liter (SOD1noCys) or 8.64 to 3.11 g/liter (SOD1(57/146)) in 50 mM Tris, 100 mM NaCl, 5 mM EDTA at pH 7.0. To remove any oligomeric/aggregated species, the E,E-SOD1noCys samples were first loaded on the gel filtration column (G2000SW, TOSOH) equilibrated with 50 mM Tris, 100 mM NaCl, 5 mM EDTA at pH 7.0, and protein fractions of a symmetrical and sharp elution peak was collected and immediately used for SAXS measurements. 10,000 hypothetical rigid-body models were randomly generated, from which a plausible ensemble was suggested by refining populations and compositions of the hypothetical structures

Results and Discussion

Mr from Vpf

Number of Shannon channels