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

Abstract

Copper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O₂). 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 SOD1·CCS1 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 SOD1·CCS1 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."

Keywords: CCS1; chaperone; copper transport; copper-zinc superoxide dismutase (SOD1); dual chaperone; enzyme maturation; metal ion-protein interaction; metallo-chaperone; metallo-protein; multifunctional enzyme; oxidative stress; post-translational modification (PTM); protein complex; superoxide dismutase (SOD).

Figure 1.

Immature SOD1 binding and activation by CCS1 D3 variants. A, fluorescence-based binding studies for immature SOD1 (E,Zn-SOD1^SH) and D3 variant forms of CCS1. Both W222A and W237A mutations cause ∼4-fold decrease in binding affinity compared with WT CCS1. Curves are normalized for comparison. B, in vitro SOD1 activity assay. SOD1 activity is shown as white bands on the black background. Lane 1, E,Zn-SOD1^SH; lane 2, E,Zn-SOD1^SH with Cu(I)-WT CCS1; lane 3, E,Zn-SOD1^SH with Cu(I)-W222A CCS1; lane 4, E,Zn-SOD1^SH with Cu(I)-W237A CCS1; lane 5, H46R/H48Q/C146S SOD1 (X,Zn-SOD1^X, cannot bind copper or form a disulfide bond) with Cu(I)-WT CCS1. Below the activation gel are Coomassie-stained loading controls for the SOD1 and CCS1 proteins.

Figure 2.

CCS1 D3 coordinates Cu(I) before delivery to SOD1. SDS-PAGE–based disulfide cross-linking analysis of tCCS1 is shown. The top four gels are for the WT and cysteine variant forms of tCCS1 and are all set up in the same reaction order. Lane 1, purified tCCS1 without βME; lane 2, tCCS1 + 10 mm βME; lane 3, tCCS1 + 25 μm CuP/no βME; lane 4, tCCS1 + 25 μm CuP + 10 mm βME. tCCS1 monomer is the faster-running (bottom band), and the disulfide cross-linked dimers are the top band. The bottom gel examines the consequence of Cu(I) addition to tCCS1. Lane 1, tCCS1 without βME; lane 2, tCCS1 + 10 mm βME; lane 3, tCCS1 + 25 μm CuP/no βME; lane 4, tCCS1 + 25 μm CuP + 10 mm βME; lane 5, Cu(I)-tCCS1 without βME; lane 6, Cu(I)-tCCS1 + 10 mm βME; lane 7, Cu(I)-CCS1 + 25 μm CuP/no βME; lane 8, Cu(I)-CCS1 + 25 μm CuP + 10 mm βME. The SOD1 activation gel shows that Cu(I)-tCCS1 can activate the yeast SOD1 (lane 3) to levels similar to that of yeast CCS1 (lane 2). Lane 1, immature yeast SOD1 alone. Below the activation gel are Coomassie-stained loading controls for the SOD1 and CCS1 proteins.

Figure 3.

Electronic absorption analysis of Cu(I) transfer pathway between SOD1 and CCS1. UV-visible absorption spectra of apo (metal-free) and Cu(I)-bound CCS1 and the absorbance shifts upon reaction with H46R/H48Q (X,Zn-SOD1^SH) and WT E,Zn-SOD1^SH are shown. In the inset, the differential absorption metal-induced contributions have been derived by subtracting the relevant apoprotein spectra from the corresponding spectra recorded on the Cu(I)-bound transfer reaction products.

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 μm) with equal volumes of apo H46R/H48Q (X,Zn-SOD1^SH) or WT E,Zn-SOD1^SH (20 μm) 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 SOD1·CCS1 2Cys/1His entry site.

Figure 5.

Zinc competition between SOD1 and the Zn(II)-chelator TPEN. Immature forms of SOD1 (E,Zn-SOD1^SH) can be readily outcompeted for zinc by the Zn(II) chelator TPEN. SOD1 maturation events (i.e. copper binding and disulfide formation) increase SOD1 affinity for zinc, as seen by the decrease in Zn(II) loss for Cu,Zn-SOD1^SS. CCS1 binding alone (i.e. apo-CCS1 with no copper delivery) promotes zinc binding by immature SOD1 (which reduces Zn(II) loss). The removal of key tryptophan residues within CCS1 D3 (Trp-222 and Trp-237) eliminates this zinc protection ability of CCS1. Error bars, S.E.

Figure 6.

Copper-dependent models for SOD1 activation by yeast CCS1. A, CCS1 domains are shown as a blue oval (D1), gray cylinder(D2), and red strand (D3). Under copper-limiting conditions, Cu(I) (blue circle) is first acquired by D1, and binding stabilizes an unfolded D3. B, CCS1 binding to immature SOD1 (green β-barrel, purple “disulfide loop,” and orange electrostatic loop) orders the conserved loop elements of SOD1 and promotes proper Zn(II) (orange circle) binding. C, Cu(I) is bound at the entry site, promoting sulfenylation at a nearby cysteine in SOD1. G, disulfide bond formation in SOD1 keys Cu(I) release to the active site, terminates interaction with CCS1, and promotes homodimerization with another mature SOD1 molecule. D and E, when labile copper is abundant, Cu(I) may be provided to the entry site by GSH after SOD1·apo-CCS1 complex formation. F, Cu(I) is bound at the entry site promoting sulfenylation at a nearby cysteine in SOD1. G, mature Sod1 homodimerizes, and CCS is free to proceed the same as before.