Protein-protein interactions involved in copper homeostasis, oxidative aging and neurodegenerative disease

Authors: Fernanda A. Sala (University of Sao Paulo)
Co-authored by industrial partner: No

Type: Thesis

State: Published (Approved)
Published: May 2020

Abstract: Despite knowledge of human Cu,Zn Superoxide dismutase (hSOD1) importance in many cellular processes, the final stages of hCCS (human copper chaperone for Superoxide dismutase 1) dependent hSOD1 maturation, have never been fully elucidated. This process involves both copper transfer to hSOD1 and disulfide oxidation and understanding it can be critical in the fight against neurodegenerative diseases, as immature forms of SOD1 are typically identified as more prone to aggregation. The main difficulties are in obtaining the transient human heterocomplexes and the behaviour of full length hCCS which is recalcitrant to crystallization due to its highly flexible structure. The objective of this work was to investigate the recognition processes and sequence of activation events during the maturation of hSOD1 catalyzed by hCCS. Protein-membrane association experiments on different combinations of hCCS and SOD1 mutants suggested that copper acquisition occurs via hCCS, in its homodimeric state, engaging with the lipid bilayer and subsequently forming heterodimers with hSOD1 off the membrane. Additionally, complex stability experiments on combinations of hCCS and hSOD1 mutants confirmed that disulphide formation seems to be both the last step in the hSOD1 maturation and the regulator of complex formation with hCCS. Based on this, we chose a set of complexes suitable for crystallographic assays. Heterodimer crystals were obtained using commercial screens and optimized using different techniques. Crystals of heterodimeric complexes which were either hSOD1 apo or holo for copper in complex with hCCS were obtained, processed and analyzed. The analysis of the structures of the heterodimeric complexes showed a novel conformation for the SOD1 disulphide sub-loop which regulates complex formation and dissociation, communicates the presence of hCCS to the hSOD1 active site and coordinates the timing of copper transfer prior to the disulphide bond formation, minimizing the production of potentially toxic hSOD1 species. Once the disulphide bridge is formed, we suggest that the hCCS Ala231 methyl dictates the heterodimer dissociation by steric effects at the interface. As a consequence, hSOD1 (in its copper loaded and disulphide oxidized form) dimerizes to mitigate this repulsion and thereby generates the mature and catalytically active homodimeric species.

Journal Keywords: ALS, protein crystallography; hCCS; heterodimer; hSOD1; post-translational modifications

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 03/06/2020 15:43

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