Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant:  The Influence of Dimerization on Mobility and Function

Banci, Lucia; Bertini, Ivano; Cramaro, Fiorenza; R, Del Conte; Rosato, Antonio; Ms, Viezzoli

doi:10.1021/bi000067z

Cited by 61 publications

(111 citation statements)

References 61 publications

(199 reference statements)

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“…This value compares with 8.4 Ϯ 0.3 ns found for the Cu-free monomeric HSOD (30), which comprises 153 residues with respect to 162 of the present construct. It is also consistent with the values found for proteins of similar size (31,32). Addition of 1.0 equivalent of Zn 2ϩ to Zn BsSOD does not produce any significant aggregation of the protein, as indicated by the reorientational correlation time equal to 10.5 Ϯ 1.1 ns, identical to that in absence of exogenous Zn, which is 10.0 Ϯ 0.7 ns.…”

Section: Resultssupporting

confidence: 89%

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Banci

Bertini

Calderone

et al. 2005

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

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Little is known about prokaryotic homologs of Cu,Zn superoxide dismutase (SOD), an enzyme highly conserved among eukaryotic species. In 138 Archaea and Bacteria genomes, 57 of these putative homologs were found, 11 of which lack at least one of the metal ligands. Both the solution and the crystal structures of the SOD-like protein from Bacillus subtilis, lacking two Cu ligands and found to be enzymatically inactive, were determined. In solution, the protein is monomeric. The available nuclear Overhauser effects, together with chemical-shift index values, allowed us to define and to recognize the typical Cu,Zn SOD Greek ␤-barrel but with largely unstructured loops (which, therefore, sample a wide range of conformations). On the contrary, in the crystal structure (obtained in the presence of slight excess of Zn), the protein is well structured and organized in covalent dimers held by a symmetric bridge consisting of a Zn ion bound to an Asp-His dyad in a tetrahedral geometry. Couples of dimers held by hydrophobic interactions and H bonds are further organized in long chains. The order͞disorder transition is discussed in terms of metal binding and physical state.conformational mobility ͉ NMR structure determination ͉ Zn-mediated protein dimers ͉ x-ray crystallography ͉ superoxide dismutase T he availability of the complete sequence of an increasing number of genomes allows comparative analyses aimed at the identification of genes and of corresponding physiological functions conserved along evolution. Cu,Zn superoxide dismutase (SOD) is a ubiquitous family of enzymes that efficiently catalyze the dismutation of superoxide anions into oxygen and hydrogen peroxide (1, 2). The protein takes a classical Greek key ␤-barrel fold and adopts a dimeric quaternary structure (3). It binds one Zn and one Cu ion per subunit, the latter ion being the site of the enzymatic reaction, whose rate limiting step is the diffusion of superoxide through the channel that leads to the Cu site (4). SOD has been considered for a long time to be peculiar of eukaryotic organisms, in which its evolutionary rate has been studied (5). From the first description of a SOD activity in Escherichia coli (6), other putative homologous of the eukaryotic protein have been then found in the periplasmic space of other bacterial species (7) The x-ray structures have been solved for the proteins from Photobacterium leiognathi (8), E. coli (9), Salmonella typhimurium (10), and Actinobacillus pleuropneumoniae (11), all of them maintaining the typical metal sites of Cu,Zn eukaryotic SODs and normal SOD activity. With the aim of providing a more detailed picture of prokaryotic SODs, we browsed the available complete genomes of Archea and Bacteria to search for sequences with some degree of homology with human SOD (HSOD), which is found in 48 of the 138 prokaryotes with complete genome. In some of them, not all of the metal ligands are present. The SOD-like protein from Bacillus subtilis (BsSOD) lacks two binding-Cu residues (His-48 and His-63, human protein numbe...

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Section: Resultssupporting

confidence: 89%

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Banci

Bertini

Calderone

et al. 2005

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

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“…We therefore hypothesize a mechanism of aggregation in which the SOD1 apoprotein, which is known to be destabilized and highly flexible because of the lack of metal ions, can sample a broad range of conformations, some of which cause the two free cysteines to be exposed to the solvent. Indeed, it has been shown previously in an engineered SOD1 monomer, artificially stabilized through two mutations at the subunit-subunit interface, that the absence of metals, and particularly of the structural metal zinc, causes the protein to be quite mobile and that several loops, including loop IV and the electrostatic loop VII, are characterized by a molten globule state within which a quite broad range of conformations are sampled (25). This flexibility is expected to make the two free cysteines more solventaccessible relative to the dimeric, metallated form.…”

Section: Discussionmentioning

confidence: 99%

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Banci

Bertini

Durazo³

et al. 2007

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

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of Ϸ650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 M protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.amyloid ͉ neurodegeneration ͉ protein aggregation ͉ amyotrophic lateral sclerosis ͉ protein misfolding P rotein oligomerization, aggregation, and formation of insoluble amyloid deposits commonly are observed in neurodegenerative diseases, but the factors initiating and modulating the abnormal protein-protein interactions that lead to oligomerization remain elusive (1, 2). Metal ions frequently have been implicated in these phenomena, but how exactly they are involved remains unclear (3). Over 114 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD1) have been linked to the neurodegenerative disease familial ALS (FALS) by a gain-of-function mechanism (4-6). Although the exact cellular sites and mechanisms of toxicity are unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (7,8). Several recent publications have presented compelling evidence that abnormal disulfide cross-linking of ALS-mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected in neural tissues of SOD1-ALS transgenic mice that are presumed to be components of higher-molecular-weight species or intermediates in their formation (7, 9-11).Wild-type (WT) human SOD1 is an exceptionally stable protein in its holo form and, although some of the ALS-mutant SOD1 proteins are severely destabilized by their mutations, others largely retain the stability of WT SOD1 (4). In the fully demetallated (apo) states, some ...

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“…In addition, the backbone oxygen atoms of residues Cys-57 and Gly-61 in this loop form three hydrogen bonds that correctly orient the side chain of Arg-143, an important determinant of copper ion accessibility in SOD1 (27,28,(45)(46)(47)(48). These structural features increase the likelihood that disulfide reduction at Cys-57, and the disorder of this loop could facilitate partial unfolding, monomerization, metal ion loss, or altered reactivity of bound copper.…”

Section: Discussionmentioning

confidence: 99%

Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

Tiwari

Hayward

2003

Journal of Biological Chemistry

202

192

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We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the ␤-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity.Amyotrophic lateral sclerosis (ALS) 1 is an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem, and brain (1). Approximately 10% of ALS cases are familial, and ϳ20% of these individuals inherit one of Ͼ90 autosomal dominant mutations in the gene encoding copper/ zinc superoxide dismutase 1 (SOD1) (2). 2 SOD1 is a 32-kDa homodimeric enzyme expressed predominantly in the cytosol that decreases the intracellular concentration of superoxide radicals (O 2 . ) by catalyzing their dismutation to O 2 and H 2 O 2 . ALS-associated mutations of conserved residues throughout the protein impart a toxic property to the enzyme that appears unrelated to its normal dismutase activity (reviewed in Ref.3). Whereas transgenic mice that overexpress mutant SOD1s consistently develop lethal motor neuron degeneration (4 -8), mice that overexpress the wild type (WT) enzyme exhibit only subtle motor abnormalities (9). In addition, SOD1 knock-out mice are not susceptible to motor neuron loss unless following axonal injury (10). Mutant SOD1 enzymes have been proposed to facilitate aberrant copper-mediated chemistry, disrupt protein recycling or chaperone function, form toxic aggregates, or induce organelle dysfunction or apoptosis (3,11,12), but the precise mechanism of specific motor neuron toxicity has not been elucidated. The observation that some mutant SOD1s exhibit accelerated turnover in vivo or increased proteolytic susceptibility compared with the WT enzyme (13-15) suggests that biologically significant perturbations of mutant SOD1 conformation occur. The induction of chaperone proteins that can protect cultured motor neurons from mutant SOD1 toxicity (16) and appear to associate with SOD1 mutants (17) provides further...

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Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function

Cited by 61 publications

References 61 publications

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

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