Wednesday, May 14, 2025
5/14/2025
Molecular Mechanisms of Sod1 Maturation Processes - Molecular Mechanisms of Sod1 Maturation Processes Copper ions are tightly regulated by aerobic organisms to the extent that labile copper is virtually undetectable. Although elevated levels are toxic, the capacity to interconvert between oxidation states (e.g. Cu(I) and Cu(II)) makes copper ions useful for a wide variety of cellular processes. Copper (and other redox-active metal ions) is constantly guarded from promiscuous reactions in the cytosol by a diverse group of sequestering agents that include trafficking proteins and molecular scavengers. Metallo-chaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. One such target is the ubiquitous antioxidant enzyme copper-zinc superoxide dismutase (Sod1). The copper chaperone for Sod1 (Ccs1) delivers a single copper ion to the active-site and catalyzes oxidation of the disulfide bond within Sod1 through a mechanistically ambiguous process. These Ccs1-mediated posttranslational modifications transform immature Sod1 from a collection of marginally stable, inactive monomers into a remarkably stable, active homodimer. Mutations in the gene that code for Sod1 have been implicated in the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Interestingly, it is the immature forms of these pathogenic Sod1 variants that make up the disease related aggregates found in susceptible cells. We have determined the structure of full-length Ccs1 bound to an immature form of Sod1 at 2.35 Å. The structure of the heterocomplex reveals a previously unobserved β-hairpin conformation of the Ccs1 C-terminal domain (D3) suggestive of a “pivot and release” mechanism for Ccs1 action. We suggest that Ccs1 binding to an immature form of Sod1 induces movement of a conserved loop element in Sod1 that exposes an electropositive hole and proposed “entry site” for copper ion delivery to Sod1. We have since our current Sod1•Ccs1 heterocomplex structure for a structure-based approach to investigate Ccs1 action using the following methods (I) a fluorescence based system designed to delineate the thermodynamics guiding Ccs1-mediated maturation of wild-type and mutant forms of Sod1, (II) structural characterization of additional Sod1•Ccs1 heterocomplexes at progressive steps in the maturation process, (III) X-ray absorption spectroscopy and related techniques to probe copper ion coordination at the Sod1 “entry site” and (IV) cell-based biochemical analysis focusing on connecting catalytic turnover at the Sod1 “entry site” and a sulfenic acid intermediate to disulfide bond formation and release of the copper ion to the Sod1 active site. The aims presented here are designed to end the ongoing debate regarding a universal mechanism for Sod1 maturation via Ccs1 and may help to illustrate the initial stages of the Sod1-linked neurodegenerative disorder ALS.
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