The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation

Abstract

Edited by Ruma BanerjeeCopper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O 2 ). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1⅐CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1⅐CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."Copper (Cu) 2 is required for the activation of dioxygen, a function essential for the survival of aerobic organisms (1). Figure 4. Kinetic analysis of CCS1 mediated copper delivery to sites on SOD1. Stopped-flow kinetic traces at 260 nm and corresponding curve fittings (red line) obtained upon rapid mixing of Cu(I)-Ccs (20 M) with equal volumes of apo H46R/H48Q (X,Zn-SOD1 SH ) or WT E,Zn-SOD1 SH (20 M) under aerobic or anaerobic conditions are shown. The decreased absorbance at 260 nm (top left) corresponds to the copper ion moving from the cysteine coordination of CCS1 and/or SOD1 and entering the histidine coordination of the SOD1 active site. Increased absorbance at 260 nm shows the copper ion transfer from CCS1 cysteines to the SOD1⅐CCS1 2Cys/1His entry site.

Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs)

“…Ccs binding weakens the affinity of the copper/active site held zinc by nearly 3-fold, while dramatically decreasing the total amount of zinc loading into the copper/active site by~60%. These results suggest that Ccs interaction promotes "site-specific" zinc binding in Sod1 by discouraging mis-metallated zinc binding in the copper/active site [16].…”

“…In this work, we aim to analyze this effect by examining the zinc affinity of these mutations in various stages of maturation. The role of Ccs in facilitating zinc acquisition by Sod1 has been recently published [16]. Here, we further elucidate the changes caused in this interaction by fALS mutations.…”

“…The immature H80R protein bound zinc with an affinity of 2.78 ± 1.30 × 10 −16 M ( Table 1). This value is~32-fold weaker than the zinc binding affinity for the "zinc-site" in wild-type Sod1 and over four orders of magnitude less than the affinity for Cu(I) at this site [16]. Notably, zinc loading into the active site was nearly saturated in the samples, with~94% of the copper/active sites occupied with zinc (measured by ICP-MS after metal reconstitution and exhaustive washing and listed in Table 1).…”

“…The human form of Ccs binds zinc in its Sod1-like domain 2 (D2) coordinated by residues homologous to those found in the Sod1 β-barrel [8]. We have previously shown that Ccs preferentially binds to a completely immature form of Sod1 (E,E-Sod1 SH ) [13] and others have suggested that Ccs may act directly or indirectly to deliver zinc to immature Sod1 [16,41]. Therefore, we wanted to measure the affinity of the Ccs D2 binding site for zinc and expected that the similar nature of Ccs and Sod1 zinc-binding sites would result in similar affinities.…”

“…Zinc is bound in a nearby site by three histidine residues (H63, H71, H80) and a single aspartate (D83). H63 "bridges" the copper and zinc ions, easing the transition between Cu(I) and Cu(II),thereby increasing enzymatic activity 10-fold [6,16].…”