Selenocysteine Positional Variants Reveal Contributions to Copper Binding from Cysteine Residues in Domains 2 and 3 of Human Copper Chaperone for Superoxide Dismutase

Abstract

The human copper chaperone for superoxide dismutase binds copper both in an Atx1-like MTCQSC motif in domain 1, and via a multinuclear cluster formed by two CXC motifs at the D3 dimer interface. The composition of the Cu(I) cluster has been investigated previously by mutagenesis of the CXC motif, and by construction of a CXU selenocysteine derivative which has permitted XAS studies at both Cu and Se absorption edges. Here we report the semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA and 243-UAUA in the D1 double mutant (C22AC25A) background, prepared by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation. By varying the position of the Se atom in the CXC motif, we have been able to show that Se is always bridging (2 Se-Cu) rather than terminal (1 Se-Cu). Substitution of both D3 Cys residues by Sec in the UAUA variant does not eliminate the Cu-S contribution, confirming our previous description of the cluster as most likely a Cu4S6 species, and suggesting that D2 Cys residues contribute to the cluster. As predicted by this model, when Cys residues C141, C144 and C227 are mutated to alanine either individually or together as a triple mutant, the cluster nuclearity is dramatically attenuated. These data suggest that Cys residues in D2 of hCCS are involved in the formation, stability and redox potential of the D3 cluster. The significance of these finding to the SOD1 thiol/disulfide oxidase activity are discussed in terms of a model in which a similar multinuclear cluster may form in the CCS-SOD heterodimer.

Interactions of Cu(I) with Selenium-Containing Amino Acids Determined by NMR, XAS, and DFT Studies

“…In proteins, Cu(I)-Se binding to incorporated selenocysteine residues has also been well characterized. 50,62-64 …”

Cellular copper distribution: a mechanistic systems biology approach

“…Copper coordination in full-length CCS is still quite controversial as, depending on the experimental conditions, variable amounts of copper ions are bound in both the yeast and human CCS proteins. Extensive EXAFS experiments have recently led to the proposal that human CCS can form two distinct clusters with different stoichiometry: a polynuclear Cu 4 S 6 cluster involving extra Cys residues from domain II and a dinuclear Cu 2 S 4 cluster when these extra Cys residues are unavailable [138,139]. The functional significance of this complex copper(I) coordination in CCS is still elusive.…”

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

“…Compounding the issue, transfer complexes are usually in the Cu(I) state which is invisible to many spectroscopic techniques. We have developed an alternative approach via the use of selenocysteine (SeC) 28 , 29 or selenomethionine (SeM) 17 , 30 labeling of the Cu(I)-coordinating Cys or Met ligands in one member of the donor−acceptor pair, where the unique Se-Cu feature in the Se K-edge extended X-ray absorption fine structure (EXAFS) spectrum is used to follow metal transfer into or out of the Se environment. The utility of the method has already been proven in a number of reports leading to proposals for the individual roles of the protein components of the cus transporter 18 , 30 , 31 .…”