A selenocysteine variant of the human copper chaperone for superoxide dismutase. A Se-XAS probe of cluster composition at the domain 3-domain 3 dimer interface.
We report the semisynthesis of a selenocysteine (Sec) derivative of the human copper chaperone for superoxide dismutase, substituted with Sec at the C-terminal C246 residue. Measurements of hCCS-induced SOD1 activation were used to show that the C-terminal CXC sequence is both necessary and sufficient for EZn-SOD maturation. Therefore, an active CAU variant carrying Sec as the terminal amino acid was prepared by expressed protein ligation of a single selenocysteine amino acid to a 243-CA truncation. This reaction proceeded in high yield and generated the desired 243-CAX (X = C or U) protein with the expected mass. Se-edge XAS of the apoprotein indicated that both Se-S and Se-Se interactions were present in a 0.3:0.7 ratio, indicating an equilibrium between species with either a selenosulfide or a diselenide cross-link. After reduction on immobilized TCEP, the ligated Cys and Sec apoproteins bound up to 2.5 Cu(I) ions per hCCS monomer with both Cu and Se as constituent atoms of the cluster which forms at the domain 3 interface of a hCCS dimer. Merging of XAS data at the Cu and Se K-absorption edges provided additional details of the cluster composition, specifically the fact that both Se atoms occupied bridging positions between two Cu(I) atoms. Further, the requirement for identical Cu-Se bond lengths and Debye-Waller factors at each absorption edge allowed us to rule out simple models for the cluster composition such as a bis-Cys(Sec)-bridged dinuclear cluster and was indicative of a more complex cluster with a nuclearity of >or=3.
Barry
Amanda N
AN
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Sciences University, Beaverton, Oregon 97006-8921, USA.
Blackburn
Ninian J
NJ
D000042:Absorption; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003300:Copper / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017279:Selenocysteine / Q000378:metabolism*; D017384:Sequence Deletion; D013057:Spectrum Analysis; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D009626:Terminology as Topic; D014965:X-Rays
C114678:CCS protein, human; D008024:Ligands; D018832:Molecular Chaperones; C000606290:SOD1 protein, human; D017279:Selenocysteine; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1