Variable Metallation of Human Superoxide Dismutase: Atomic Resolution Crystal Structures of Cu–Zn, Zn–Zn and As-isolated Wild-type Enzymes

doi:10.1016/j.jmb.2005.11.081

Journal of Molecular Biology

Volume 356, Issue 5, 10 March 2006, Pages 1152-1162

https://doi.org/10.1016/j.jmb.2005.11.081 Get rights and content

Human Cu–Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn–Zn, Cu–Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn–Zn structure appears to be at least as stable as the correctly (Cu–Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn–Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.

Introduction

Human superoxide dismutase (SOD1) performs an important role as an antioxidant enzyme in almost all cells of the human body, its principal catalytic action being the dismutation of the superoxide anion, a normal product of cell metabolism, to hydrogen peroxide and dioxygen. Each subunit of the homodimeric SOD1 contains one Cu atom, which is the site of catalytic activity, and one Zn atom, which has long been thought to have a role in providing the necessary structural framework. Mutations in the SOD1 gene have been implicated as a cause of the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (FALS), also known as motor neuron disease or Lou Gehrig's disease.1, 2 Although the mechanism of SOD1-induced FALS is not known, there is a growing body of evidence to suggest that it involves a “gain-of-function” of the mutant protein rather than a simple misfunction of SOD1, such as lower catalytic activity brought about by the mutation.3, 4, 5 Protein misfolding and aggregation has been implicated strongly in the gain-of function toxicity of SOD1 mutants. The fact that the Cu atom is known to react with hydrogen peroxide or peroxynitrite has led to the suggestion that oxidative damage caused by these reactions, either to the mutant protein itself or by generation of radical species that diffuse away from SOD1 and attack cellular components, may be involved in the SOD1 FALS pathogenesis.4, 5 The presence of Cu is clearly essential for the oxidative mechanisms to apply, and there is strong evidence that the extent of both Cu and Zn metallation of the enzyme is a key factor in understanding how oxidative damage, dimer destabilisation and aggregation of mutant SOD1 cause FALS.6, 7, 8, 9, 10, 11, 12

The thermodynamic and kinetic stability of wild-type and mutant enzymes, and the monomer–dimer equilibrium of SOD1 are also significantly dependent upon the presence of metals.9, 10, 13, 14, 15, 16 The metal atoms have an essential role in enabling the formation of stable and well-ordered active site, electrostatic and Zn binding loop regions. Structural data from X-ray crystallography have shown that in the metal-deficient FALS SOD1 mutants apo-S134N, apo-H46R and Zn-H46R, these loops are disordered, leading the SOD1 functional dimers to combine, producing amyloid-like linear or helical filaments.17, 18 Significantly, the X-ray data for metal-deficient wild-type SOD1 (containing ∼20% Zn in the Zn-binding sites) also showed evidence for the existence of amyloid-like structures, with near-linear zig–zag filaments being formed from alternating ordered–disordered dimers.¹⁹ It is possible that metallation, oxidative damage, and aggregation work together to produce the toxic properties of FALS SOD1 mutants: damage to the metal-binding sites of mutant SOD1 by hydrogen peroxide may cause loss of metal and promote protein–protein aggregation.⁵

In view of these observations, the variability of metal loading in wild-type as well as mutant SOD1 is of considerable interest. Here, we present three “atomic-resolution” crystal structures of wild-type SOD1. In each of these structures, the Zn-binding site was correctly metallated with Zn but the Cu-binding site had varied occupancy. In the as-isolated recombinant enzyme, which was expressed in yeast cells, the 1.24 Å resolution structure showed that the Cu-binding site contained a mixture of Cu and Zn atoms. Recombinant apo-enzyme, re-metallated with Zn atoms only, gave a Zn–Zn SOD1 structure that was also solved to 1.24 Å resolution. The native human enzyme was reconstituted with copper to give an enzyme containing only Cu atoms at the Cu-binding site (i.e. a fully Cu–Zn enzyme). This structure was determined to 1.07 Å resolution and is the only atomic-resolution structure for human SOD1 and the first structure of human SOD in the reduced, Cu(I) form. The presence of Cu or Zn in the Cu binding site was confirmed by anomalous X-ray scattering measurements and the oxidation state of Cu in the crystals was confirmed by X-ray absorption edge data.

Section snippets

Results and Discussion

Recent crystal structures of recombinant wild-type human SOD1, expressed in Saccharomyces cerevisiae cells, include the fully metallated enzyme at 1.78 Å resolution and a Zn-deficient apo form at 1.82 Å resolution.¹⁹ In addition, the structure of the wild-type enzyme expressed in Escherichia coli cells was published at 1.70 Å resolution.²⁰ The structures reported here provide significant improvements in resolution, accuracy, and precision for the structure of the wild-type enzyme, under conditions

Conclusions

The structures of as-isolated, Zn–Zn and Cu–Zn SOD1 have been solved to 1.24 Å, 1.24 Å and 1.07 Å, respectively. The latter is the first atomic-resolution structure for intact human SOD1 and the first structure of reduced, Cu(I) human SOD1. The Zn–Zn structure is the first to show this metallation state. The observation of Zn bound at the Cu site in recombinantly expressed SOD1 indicates that, in the absence of the CCS chaperone and subsequent conditions of low copper availability, the Cu site may

Crystallisation

Recombinant human SOD1 protein was expressed and purified as described previously.¹⁰ All crystals were grown using the sitting-drop, vapour-diffusion method. As-isolated and Zn–Zn recombinant protein were both crystallised using 2.5 M ammonium sulphate as precipitant with 100 mM NaCl in 100 mM Tris–HCl buffer (pH 7.5). Crystal dimensions were 1.0 mm×1.0 mm×0.1 mm (as-isolated enzyme) and 0.1 mm×0.1 mm×0.05 mm (Zn–Zn enzyme). Reconstituted Cu–Zn crystals (dimensions 0.2 mm×0.2 mm×0.1 mm) were grown using 3 M

Acknowledgements

This work was supported by the Motor Neuron Disease Association, U.K. (to S.S.H.) and the National Institutes of Health (grant GM28222, to J.S.V.) and the ALS Association (to J.S.V.). We thank our colleagues in the International Consortium on SOD and ALS (ICOSA) for valuable discussions. The authors thank CCLRC for provision of facilities at Daresbury Laboratory. We thank the UK-CRG at ESRF (MAD beamline BM-14) for provision of time. We are very grateful to BBSRC, MRC, EPSRC, NWDA and CCLRC for

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Journal of Molecular Biology

Variable Metallation of Human Superoxide Dismutase: Atomic Resolution Crystal Structures of Cu–Zn, Zn–Zn and As-isolated Wild-type Enzymes

Introduction

Section snippets

Results and Discussion

Conclusions

Crystallisation

Acknowledgements

J. Biol. Chem.

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Structure

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J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Methods Enzymol.

Mutations in Cu/Zn Superoxide-dismutase gene are associated with familial amyotrophic-lateral-sclerosis

Nature

Copper–zinc superoxide dismutase and amyotrophic lateral sclerosis

Annu. Rev. Biochem.

From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS

Nature Rev. Neurosci.