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

Stefanie D Boyd,Jenifer S Calvo,Li Liu,Morgan S Ullrich,Amélie Skopp,Gabriele Meloni,Duane D Winkler

doi:10.1074/jbc.ra118.005283

Stefanie D Boyd, Jenifer S Calvo + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra118.005283

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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."

Highlights

Copper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O2)
Another conserved tryptophan residue (Trp-237) packs into a hydrophobic pocket on D2 and essentially works as a “latch” stabilizing the compact positioning of the D3 CXC motif near the SOD11⁄7CCS1 interface
We propose a model in which Cu chaperone for SOD1 (CCS1)-mediated SOD1 activation involves Cu(I) transfer from CCS1 to SOD1 through a Cu ion entry site form where it is subsequently shuttled to the SOD1 active site

Summary

The abbreviations used are

Copper; Zn, zinc; LMCT, ligand-to-metal charge transfer; ICP-MS, inductively coupled plasma-MS; TPEN, N,N,NЈ,NЈtetrakis(2-pyridinylmethyl)-1,2-ethanediamine; TCEP, tris(2-carboxyethyl). The second domain (D2, residues 78 –216) possesses sequence and structural homology with SOD1 [21], suggesting that it recognizes SOD1 by mimicking SOD11⁄7SOD1 homodimeric interactions Supporting this notion, phosphine; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane1,3-diol; ␤ME, ␤-mercaptoethanol; BCS, bathocuproine sulfonate; TEV, tobacco etch virus; CuP, copper 1,10-phenanthroline. Our recently published SOD11⁄7CCS1 heterocomplex structure reveals a previously unobserved ␤-hairpin conformation of D3 that is stabilized by interactions with residues from SOD1, CCS1 D2, and the linker region between D1 and D2 [25] This novel conformer places the conserved CXC motif near the heterodimeric interface and creates an entry site for Cu delivery during SOD1 activation [25]. A detailed biochemical evaluation of these newly discovered molecular chaperoning functions of CCS1 is severely lacking

Results

Discussion

Experimental procedures