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Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):E4568-76. doi: 10.1073/pnas.1308531111. Epub 2014 Oct 14.

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes.

Author information

1
Department of Integrative Structural and Computational Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; Life Sciences and.
2
Department of Chemistry and Chemical Biology and.
3
Physical Biosciences Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
4
Department of Biochemistry, University of Georgia, Athens, GA 30602.
5
Center for Advanced ESR Studies (ACERT), Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301; and.
6
Department of Integrative Structural and Computational Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; Life Sciences and jat@scripps.edu edg@scripps.edu.
7
Department of Integrative Structural and Computational Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; jat@scripps.edu edg@scripps.edu.

Abstract

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.

KEYWORDS:

ESR spectroscopy; Lou Gehrig’s disease; protein aggregation; protein conformation; small-angle X-ray scattering

PMID:
25316790
PMCID:
PMC4217430
DOI:
10.1073/pnas.1308531111
[Indexed for MEDLINE]
Free PMC Article

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