Fig 1.
Comparison of human and yeast CCS-SOD1 complex structures.
The human CCS-SOD1 complex is presented in two conformations, elongated (6FON) and compact (6FP6) and is compared with the structure of yeast CCS-SOD1 (1JK9). Relevant domains and loops are coloured: SOD1 disulphide sub-loop, pink; CCS domain I, green; CCS C-terminal domain, mauve. For a complete breakdown of structures and mutations presented, please see S1B Fig. CCS, copper chaperone for SOD1; SOD1, superoxide dismutase-1.
Fig 2.
hCCS dimer interface destabilisation.
(A) Electron density map (2Fo-Fc, contoured at 1σ level) showing the Coulombic interaction between hCCS Arg104 and Asp136/Thr138. (B) Surface charge maps of hSOD1 and hCCS domain II dimer interface surfaces. (C) The hCCS (pink) dimer interface Arg232-Gly135 hydrogen bond is weakened by the steric effect of Ala231 side chain. The hCCS Gly135-Asp136 carbonyl rotates to accommodate the methyl group, and Gly135 is pushed away from Arg232. hSOD1 (cyan) Gly150 maximises hydrogen bond strength between Phe51 and Ile151. (D) Restoring SOD1-like dimer affinity with the Ala231Gly hCCS mutation vastly slows complexation. (E) Eukaryotic CCS sequence diversity shows Ala231 is very highly conserved despite its detrimental effect on homodimer affinity. (F) SOD1 Gly150 is equally well conserved, indicating the relative balance of SOD1 and CCS homodimer affinities is evolutionarily static. AU, absorbance unit; CCS, copper chaperone for SOD1; hCCS, human copper chaperone for SOD1; hSOD1, human superoxide dismutase-1; SOD1, superoxide dismutase-1; wt, wild-type.
Fig 3.
hSOD1 induced-fit complexation.
(A) Complexation with hCCS forces the hSOD1 disulphide sub-loop to adopt an open conformation separating the amino acids involved in disulphide formation. (B) Interactions across the heterodimer interface stabilise the hSOD1 disulphide sub-loop. (C) The hSOD1 Arg143 side chain interleaved between the β-barrel and disulphide sub-loop, hydrogen bonded with the DNT motif Asn53, and compared with the conformation found in homodimeric hSOD1 (magenta). (D) hCCS domain III is recruited to the hSOD1 disulphide sub-loop by a restrictive domain I position and hydrogen bonding between hCCS C-terminal Asn239 and hSOD1 Thr58. This brings the functional hCCS CXC motif into the vicinity of hSOD1 Cys57. 2Fo-Fc electron density maps are contoured at the 1σ level. hSOD1 is shown in cyan, hCCS in pink. CXC, Cys-Xxx-Cys; GDNT, Gly51-Asp52-Asn53-Thr54; hCCS, human copper chaperone for SOD1; hSOD1, human copper chaperone for SOD1; SOD1, superoxide dismutase-1.
Fig 4.
SOD1 Gly51 is the fulcrum of a molecular lever.
(A) Dihedral and (B) conformational change on the formation of the SOD1 disulphide. Rotation around Gly51 pitches the loop toward the SOD1 β-barrel, forcing a steric clash with hCCS Ala231. (C) Represented schematically. hCCS, human copper chaperone for SOD1; SOD1, superoxide dismutase-1.
Fig 5.
Dissimilar CCS membrane interaction surfaces.
(A) The structure of domain II β-barrel loops I, IV, and VII are not conserved among yeast and human CCS orthologues. (B) Coulombic charge representation showing differing putative hCCS and yCCS domain II membrane-interacting surfaces. (C) Liposome-binding assay showing how the concerted effect of all three hCCS domains facilitate membrane association (S7C and S7D Fig). hCCS dimer affinity, domain I electropositivity, and complexation with SOD1 all effect lipid association. Mean ± SEM, n = 5. CCS, copper chaperone for SOD1; hCCS, human copper chaperone for SOD1; SOD1, superoxide dismutase-1; yCCS, yeast copper chaperone for SOD1.
Fig 6.
hCCS-catalysed hSOD1 activation.
1. hCCS Ala231 facilitates a dynamic association with membranes while mobilising monomeric hCCS for complexation with hSOD1. 2. Noncovalent interactions between hCCS domain II and the GDNT motif of the plastic hSOD1 disulphide sub-loop dictate molecular recognition. Complexation-induced conformational rearrangement prepares hSOD1 to receive copper, while impeding disulphide formation. 3. hSOD1 disulphide formation breaks GDNT motif interactions, rotates the SOD1 disulphide loop, and weakens heterodimer affinity. 4. hSOD1 forms a stable, active homodimer. hCCS can homodimerise and relocate back to the cytoplasmic membrane. hCCS, pink; hSOD1, blue. Panels show interactions across dimer interfaces, with disulphide status indicated as SH, reduced; S-S, intact. Ctr1, copper transporter-1; GDNT, Gly51-Asp52-Asn53-Thr54; hCCS, human copper chaperone for SOD1; hSOD1, human superoxide dismutase-1.