Molecular recognition and maturation of SOD1 by its evolutionarily destabilised cognate chaperone hCCS

Abstract

Superoxide dismutase-1 (SOD1) maturation comprises a string of posttranslational modifications which transform the nascent peptide into a stable and active enzyme. The successive folding, metal ion binding, and disulphide acquisition steps in this pathway can be catalysed through a direct interaction with the copper chaperone for SOD1 (CCS). This process confers enzymatic activity and reduces access to noncanonical, aggregation-prone states. Here, we present the functional mechanisms of human copper chaperone for SOD1 (hCCS)–catalysed SOD1 activation based on crystal structures of reaction precursors, intermediates, and products. Molecular recognition of immature SOD1 by hCCS is driven by several interface interactions, which provide an extended surface upon which SOD1 folds. Induced-fit complexation is reliant on the structural plasticity of the immature SOD1 disulphide sub-loop, a characteristic which contributes to misfolding and aggregation in neurodegenerative disease. Complexation specifically stabilises the SOD1 disulphide sub-loop, priming it and the active site for copper transfer, while delaying disulphide formation and complex dissociation. Critically, a single destabilising amino acid substitution within the hCCS interface reduces hCCS homodimer affinity, creating a pool of hCCS available to interact with immature SOD1. hCCS substrate specificity, segregation between solvent and biological membranes, and interaction transience are direct results of this substitution. In this way, hCCS-catalysed SOD1 maturation is finessed to minimise copper wastage and reduce production of potentially toxic SOD1 species.

The biophysics of superoxide dismutase-1 and amyotrophic lateral sclerosis

“…3). We have recently shown disulphide subloop plasticity to also be a necessary precursor of heterodimerisation with hCCS (Sala et al, 2019). Lyons et al (1996) showed that mature Ala4Val, Gly85Arg and Gly93Ala SOD1 are less resistant to intra-subunit disulphide reduction on exposure to ascorbate than the wild-type form.…”

Parameterization of Divalent Cations for Coarse-Grained Simulations

“…This may highlight the importance of electrostatics stabilization of metal binding sites. Another important remark is that SOD1 can also coordinate Copper within the same binding site 42 . Hence, it could be also hypothesized that one of the monomers, or the quaternary structure, presents rather subtle but important differences that are not captured by the CG approach.…”

Molecular and pharmacological chaperones for SOD1

“…Thus, the stabilising effect of complexation with hCCS permeates further than the heterodimer interface. However, the largest structural discrepancy between mature SOD1 and that found bound to hCCS is the adoption of an induced fit disulphide sub-loop conformation at the heterodimer interface [30]. Substitution of hCCS Arg232 and Arg104 for SOD1 Ile151 and Asn19, respectively, create non-covalent interactions across the heterodimer interface with the SOD1 disulphide sub-loop.…”

“…hCCS domain II binds to SOD1 through an ATP-independent mechanism bringing functional hCCS domains I and III into the proximity of nascent SOD1. Full-length and domain II truncated hCCS form heterodimeric complexes with intra-subunit disulphide bond reduced wild-type SOD1 and every ALS mutant directly tested to date [29][30][31][32]. Dissociation constants of 42, 68, 35 and 33 nM have been measured for hCCS binding to metal-free Ala4Val, His80Arg, Gly85Arg and Gly93Ala mutant SOD1, respectively [31].…”

“…Substitution of hCCS Arg232 and Arg104 for SOD1 Ile151 and Asn19, respectively, create non-covalent interactions across the heterodimer interface with the SOD1 disulphide sub-loop. A further repulsive interaction between hCCS Ala231 and SOD1 Gly51 prevents the sub-loop occupying the conformation found in the mature enzyme and also assists complex dissociation once PTM events are complete [30]. These interactions are the source of hCCS specificity for disulphide reduced SOD1 and prime the substrate for PTMs.…”