Behind the ALS - How does the protein SOD1 convert between physiological and pathological states?

ALS is a famous neurodegenerative disease. Misfolded SOD1 has been linked to both familial and sporadic ALS. Here, we present a cryo-EM structure of SOD1 fibrils, providing insights into the conversion of SOD1 from its immature form into an aggregated form during pathogenesis of ALS.

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Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig’s disease, is a famous progressive, fatal neurodegenerative disease that involves the loss of upper and lower motor neurons1. The sod1 gene, serving as a major antioxidant gene, was the first to be linked to the familial form of ALS1. The misfolding of human Cu, Zn-superoxide dismutase (SOD1) in motor neuron cells play a crucial role in etiology of the disease1. Misfolded SOD1 aggregates were widely observed in the spinal cords of both genetic ALS and sporadic ALS cases2. The functional human SOD1 is a 32-kDa homo-dimeric metalloenzyme; each subunit consists of 153 amino acids and contains one copper ion and one zinc ion3. The SOD1 structure in each subunit features an antiparallel β-barrel composed of eight β-strands and two α-helices, which is stabilized by a disulfide bond between Cys57 and Cys146 (refs. 3). In sharp contrast, the high-resolution structures of SOD1 amyloid fibrils are not available so far1. Therefore, it is unclear for the conformational conversion of SOD1 from its immature form with no post-translational modifications into an aggregated form during pathogenesis of ALS. To address this knowledge gap, we became interested in how SOD1 converts physiological and pathological states. 

Recently, we reported the cryo-EM structures of amyloid fibrils formed by full-length human prion protein and its E196K mutation, a genetic Creutzfeldt-Jakob disease–related mutation4,5. At the start of the project, we decided to investigate whether SOD1 fibrils formed in vitro share toxic properties with ALS inclusion, and for this purpose, we prepared homogeneous amyloid fibrils from recombinant, full-length apo human SOD1 under reducing conditions. Crucially, the SOD1 fibrils exhibited cytotoxicity to both SH-SY5Y cells and HEK-293T cells in a dose-dependent manner, and caused severe mitochondrial impairment in both cell lines. This indicates that full-length apo SOD1 forms cytotoxic, mitochondrial dysfunction-inducing amyloid fibrils under reducing conditions. 

We next determined the atomic structure of the cytotoxic SOD1 amyloid fibrils by cryo-EM. We have discovered that the SOD1 fibril consists of a single protofilament with a left-handed helix. We unambiguously built a structure model of SOD1 fibril comprising the N-terminal segment (residues 3-55) and the C-terminal segment (residues 86-153) at 2.95 Å. The fibril core exhibits a serpentine fold comprising the N-terminal segment and the C-terminal segment. Interestingly, the density of an intrinsic disordered segment comprising residues 56 to 85 is invisible due to high flexibility, which is reminiscent of the internal disordered segments observed in the structures of patient-derived amyloid fibrils from systemic AL amyloidosis6

The SOD1 fibril core features a very compact fold containing thirteen β-strands (β1 to β13) and an in-register intramolecular β-strand architecture. Six β-strands (β1 to β6) and seven β-strands (β7 to β13) are present in the N- and C-terminal segments of the SOD1 fibril core structure, respectively. The SOD1 fibril contains a long intramolecular interface comprising residues 36 to 48 in the N-terminal half and residues 98 to 109 in the C-terminal half. The two segments are zipped up by three salt bridge pairs. Interestingly, side chains of most residues in the interior of the intramolecular L-shaped interface are hydrophilic. 

Finally, we have compared the structures of apo SOD1 dimer and SOD1 fibril produced under reducing conditions (Fig. 1). Crucially, the SOD1 molecule adopts largely distinctive secondary, tertiary, and quaternary structures in two different states of SOD1, highlighting the high structural polymorphs and phenotypic diversity of SOD1 in physiological and pathological states. The apo human SOD1 dimer contains eight β-strands (to form an antiparallel β-barrel), two α-helices, and a single disulfide bond between Cys57 in a1 and Cys146 in β8’ in each subunit (Fig. 1a, b). In contrast, once folding into cytotoxic, mitochondrial dysfunction-inducing fibril structure, SOD1 molecules forms six β-strands (β1 to β6) by its N-terminal segment (residues 3 to 55) and seven β-strands (β7 to β13) by its C-terminal segment (residues 86 to 153), exhibiting an in-register intramolecular β strand architecture (Fig. 1a, c). Moreover, the cytotoxic SOD1 fibril structure features a long, mostly hydrophilic intramolecular L-shaped interface and an intrinsic disordered segment comprising residues 56 to 85 (Fig. 1a, c). 

Strikingly, among two hundred and sixteen genetic mutations of SOD1 identified from different familial ALS1, one hundred and eighty-two clinically identified mutations are located within the SOD1 fibril core structure determined in this study, in which one hundred and five representative genetic ALS-related mutations are listed in Fig. 1a. Crucially, residues forming strong salt bridges (His43, His46, Glu100, Asp101, and Asp109) that contribute to the stabilization of the intramolecular L-shaped interface between the N- and C-terminal parts of SOD1 fibril or hydrogen bonds (Val14 and Asp125) that contribute to the maintenance of the SOD1 fibril structure are also ALS-associated mutation sites1. Based on our cryo-EM fibril structure, the disease mutations, such as H43R, H46R, H46D, E100K, D101G, D101N, D109Y, D109N, and D125H (Fig. 1a, salt bridge mutations, and hydrogen bond mutations), may disrupt important interactions in the cytotoxic SOD1 fibril structure. This suggests that the different mutations may induce SOD1 to form fibrils with structures and cytotoxicity distinct from the one presented here, which might be related to the structural diversity of SOD1 fibrils, strains, and phenotypic diversity of SOD1 in pathological state1.

Figure 1

Figure 1. Comparison of the structures of apo SOD1 dimer and SOD1 fibril. 

In summary, we have discovered that the SOD1 fibril displays a very compact fold with an internal disordered segment, which contains thirteen β-strands stabilized by five hydrophobic cavities and four hydrogen bonds, and a long, mostly hydrophilic intramolecular L-shaped interface stabilized by three strong salt bridges. The comparison of the structures of apo SOD1 dimer and SOD1 fibril reveals the substantial conformational conversion from a β-sheet-rich (correspond to the antiparallel β-barrel structure), immature form of SOD1 to a totally distinct β-sheet-rich (correspond to an in-register intramolecular β strand architecture), fibrillar form of SOD1 during pathogenesis of ALS. We hope that our work will serve as a resource for researchers studying the structural polymorphism of SOD1 strains and their relationship to ALS. 

References:

  1. Ayers, J. I. & Borchelt, D. R. Phenotypic diversity in ALS and the role of poly-conformational protein misfolding. Acta Neuropathol. 142, 41-55 (2021).
  2. Grad, L. I. et al. Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 111, 3620-3625 (2014).
  3. Strange, R. W. et al. The structure of holo and metal-deficient wild-type human Cu, Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis. J. Mol. Biol. 328, 877-891 (2003).
  4. Wang, L. Q. et al. Cryo-EM structure of an amyloid fibril formed by full-length human prion protein. Nat. Struct. Mol. Biol. 27, 598-602 (2020).
  5. Wang, L. Q. et al. Genetic prion disease-related mutation E196K displays a novel amyloid fibril structure revealed by cryo-EM. Sci. Adv7, eabg9676 (2021).
  6. Radamaker, L. et al. Cryo-EM reveals structural breaks in a patient-derived amyloid fibril from systemic AL amyloidosis. Nat. Commun. 12, 875 (2021).

Yi Liang

Professor of Biochemistry, Wuhan University