Binding of a Single Zinc Ion to One Subunit of Copper−Zinc Superoxide Dismutase Apoprotein Substantially Influences the Structure and Stability of the Entire Homodimeric Protein

Potter, Soshanna Zittin; Zhu, Haining; Shaw, Bryan F.; Rodríguez, Jorge A.; Doucette, P.A.; Sohn, Suk Ho; Durazo, Armando; Faull, Kym F.; Gralla, Edith Butler; Nersissian, and Aram M.; Valentine, Joan Selverstone

doi:10.1021/ja066690+

Cited by 101 publications

(102 citation statements)

References 62 publications

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“…4C, SOD1 with 1 zinc ion per dimer fibrillated with kinetics very similar to those for apo-SOD1, whereas isolated SOD1 containing zinc ions in both subunits showed a much slower rate of elongation. The absence of an effect caused by the binding of 1 zinc ion per apo-SOD1 dimer is surprising, because 1 zinc per dimer confers considerable structural stabilization in other assays (21). In contrast, the presence of 2 copper ions per dimer, either alone or in conjunction with bound zinc, completely inhibited the formation of amyloid fibrils, showing that copper-bound, disulfide-intact forms of SOD1 are not easily recruited by SOD1 amyloid nuclei.…”

Section: Resultsmentioning

confidence: 99%

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Chattopadhyay¹,

Durazo²,

Sohn³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Familial amyotrophic lateral sclerosis (fALS) caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. Similar inclusions observed in fALS transgenic mice have a fibrillar appearance suggestive of amyloid structure. Metal-free apo-SOD1 is a relatively stable protein and has been shown to form amyloid fibers in vitro only when it has been subjected to severely destabilizing conditions, such as low pH or reduction of its disulfide bonds. Here, by contrast, we show that a small amount of disulfide-reduced apo-SOD1 can rapidly initiate fibrillation of this exceptionally stable and highly structured protein under mild, physiologically accessible conditions, thus providing an unusual demonstration of a specific, physiologically relevant form of a protein acting as an initiating agent for the fibrillation of another form of the same protein. We also show that, once initiated, elongation can proceed via recruitment of either apo-or partially metallated disulfideintact SOD1 and that the presence of copper, but not zinc, ions inhibits fibrillation. Our findings provide a rare glimpse into the specific changes in a protein that can lead to nucleation and into the ability of amyloid nuclei to recruit diverse forms of the same protein into fibrils.amyloid ͉ amyotrophic lateral sclerosis ͉ neurodegeneration ͉ protein aggregation T he antioxidant metalloprotein copper-zinc superoxide dismutase (SOD1) is a 153-residue, ␤-rich, homodimeric protein that is abundantly present in the cytoplasm. Each subunit of the mature form contains a copper ion, a zinc ion, and a disulfide bond (1). In vitro studies have shown that the presence of the metal cofactors, copper and zinc, protect the disulfide bond from reduction, suggesting a possible explanation for the persistence of the disulfide bond in the reducing environment of the cytoplasm (2). More than 100 mutations in SOD1 have been linked to the familial form of amyotrophic lateral sclerosis (fALS), a fatal neurodegenerative disease caused by selective death of motor neurons. Neuronal death is attributed to a toxic gain of function by mutant SOD1, but the exact mechanism of toxicity is unknown. Mutations in SOD1 that have been identified in fALS patients occur in 74 positions that are well dispersed over the length of this 153-residue polypeptide (3).Proteinaceous aggregates have been found in the spinal cords of ALS patients, and immunomicroscopy has confirmed that the protein inclusions present in the spinal cords of SOD1-fALS patients are rich in SOD1 (4, 5). Transgenic mice expressing human SOD1 mutants that cause fALS share many symptoms with their human counterparts, including progressive motor neuron degeneration and the presence of detergent-resistant, SOD1-rich aggregates in their spinal cords (6, 7). These aggregates have recently been shown to consist primarily of full-length, unmodified SOD1 (6). The visible proteinaceous inclusions have a fibrillar appearance and bind thioflavin S, suggesting a...

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Section: Resultsmentioning

confidence: 99%

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Chattopadhyay¹,

Durazo²,

Sohn³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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205

259

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“…The effects of zinc and cobalt binding to disulfide-intact SOD1 have been well documented. Compared with apo-WT S-S , the binding of the first zinc to the dimer results in a more thermodynamically stable protein that melts at a higher temperature in DSC experiments and has a more tightly packed structure resulting in slower hydrogen-deuterium exchange (37). The binding of the second zinc raises the melting transition to even higher temperatures (37).…”

Section: Discussionmentioning

confidence: 99%

The Disulfide Bond, but Not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

Chattopadhyay

Nwadibia

Strong

et al. 2015

Journal of Biological Chemistry

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Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wildtype SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril. Familial amyotrophic lateral sclerosis (FALS)3 caused by mutations in the SOD1 gene shows similarities to other members of the large class of neurodegenerative disorders that are characterized by gradual aggregation of specific proteins and subsequent cell death in characteristic regions of the central nervous system. Some prominent examples are amyloid-␤ deposits in Alzheimer disease, ␣-synuclein and hyperphosphorylated Tau protein deposits in Parkinson disease, prion protein deposits in the transmissible spongiform encephalopathies, and huntingtin deposits in Huntington disease (1). In the case of SOD1-linked FALS, protein deposits have been identified in affected tissues from patients and from ALS-SOD1 transgenic mice consisting largely of aggregated copper,zinc superoxide dismutase protein (Cu,Zn-SOD, SOD1 protein).Our current understanding of this class of diseases is that the implicated proteins misfold, aggregate, and ultimately deposit as extracellular plaques or intracellular inclusions in the afflicted regions of the CNS. Although the exact structures contributing to these heterogeneous protein aggregates are unknown, such diseases are nevertheless usually referred to as "amyloid diseases" because fibrillar properties characteristic of cross-␤-sheet amyloid are often detected in them and because each of the isolated proteins implicated in causing disease shows a...

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“…The task of the protein matrix then would be to select the substrate electrostatically and to protect the Cu +/2+ ion from reacting with other molecules (7). Instead, the most apparent role of the SOD1 dynamics is in structural crosstalk: the coupling between barrel and loop motions explains mechanistically how fractional metallation of one monomer influences not only the binding of subsequent metal ions, but also the structure and stability of the entire homodimer (14,27,33,34). Where there are tuneable dynamics there also is allostery (4), even if it happens to pass undetected in conformationally biased X-ray structures (13).…”

Section: Discussionmentioning

confidence: 99%

“…Together, these features form an intricate network of structural communication within the SOD1 molecule, spanning from the dynamic motions of the monomeric scaffold, via metal binding and the active-site loops, to the dimer interface. As a clue to the functional role of the communication, the binding of a single Zn 2+ ion to one subunit of the dynamic apoSOD1 dimer is found to alter the structure and stability of the entire molecule (14). Based on the general view that globular proteins with compromised structural rigidity are at increased risk of misfolding (5,6), the dynamic apoSOD1 molecule also has drawn attention as a possible precursor for pathological aggregation in ALS (12,13,15).…”

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confidence: 99%

Global structural motions from the strain of a single hydrogen bond

Danielsson

Awad

Saraboji

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the β-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cubound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?D ynamic motions of folded proteins in several cases are found to be essential for allosteric control of ligand binding, adaptive orientation of active-site moieties, and communication between distant sites in protein structures and complexes (1-4). Despite this fundamental role of dynamic motions in biological function, the molecular factors that control structural fluctuations and conformational heterogeneity within folded proteins remain largely unexplored. Also, it is not yet known if there is any relation, or principal difference, between the functional motions of proteins and the supposedly adverse structural fluctuations implicated in protein misfolding and aggregation (5, 6). An interesting system for shedding more light on these questions is the enzyme superoxide dismutase 1 (SOD1) associated with pathological misfolding and aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Native SOD1 is a symmetric homodimer of two Ig-like β-barrels, each of which coordinates one catalytic Cu +/2+ ion and one structural Zn 2+ ion. The redox active Cu +/2+ ion is ligated directly to the side of the monomer β-barrel, whereas the Zn 2+ ties together the long loops IV and VII to a densely packed dome over the active site. The role of this dome is twofold. It composes a selectivity filter for substrates to the Cu +/2+ site (7) and also provides a critical part of the dimer interface (8). As a consequence, the dimerization of SOD1 becomes tightly controlled by metal binding via the structure of loops IV and VII, and the conserved C57-C146 disulfide linkage (9): if the metals and disulfide bond are lost, t...

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Binding of a Single Zinc Ion to One Subunit of Copper−Zinc Superoxide Dismutase Apoprotein Substantially Influences the Structure and Stability of the Entire Homodimeric Protein

Cited by 101 publications

References 62 publications

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

The Disulfide Bond, but Not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

Global structural motions from the strain of a single hydrogen bond

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