RESUMO
Copper chaperone for superoxide dismutase 1 (SOD1), CCS, is the physiological partner for the complex mechanism of SOD1 maturation. We report an in vitro model for human CCS-dependent SOD1 maturation based on the study of the interactions of human SOD1 (hSOD1) with full-length WT human CCS (hCCS), as well as with hCCS mutants and various truncated constructs comprising one or two of the protein's three domains. The synergy between electrospray ionization mass spectrometry (ESI-MS) and NMR is fully exploited. This is an in vitro study of this process at the molecular level. Domain 1 of hCCS is necessary to load hSOD1 with Cu(I), requiring the heterodimeric complex formation with hSOD1 fostered by the interaction with domain 2. Domain 3 is responsible for the catalytic formation of the hSOD1 Cys-57-Cys-146 disulfide bond, which involves both hCCS Cys-244 and Cys-246 via disulfide transfer.
Assuntos
Cobre/química , Liases/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/fisiologia , Sítios de Ligação , Cisteína/química , Dissulfetos/química , Humanos , Cinética , Liases/química , Espectroscopia de Ressonância Magnética/métodos , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , Superóxido Dismutase-1 , Fatores de TempoRESUMO
ATP7B is a human P(1B)-type ATPase that has a crucial role in maintaining copper(I) homeostasis. Mutations in the corresponding gene are the cause of Wilson disease. Among its various distinguishing features is a long ( approximately 630 amino acids) N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We expressed the entire tail as a single construct in Escherichia coli and investigated its interaction with its copper chaperone (i.e. HAH1) by solution NMR spectroscopy. We observed that all six of the metal-binding domains were metallated by Cu(I)-HAH1, with the first, the second, and the fourth domains forming an adduct with it. This behavior is different from that of the highly similar human ATPase ATP7A, in which only two domains form such an adduct. The distinct behaviors of the different domains were analyzed in terms of the energetics of Cu(I) transfer, hinting at a specific role of the interaction with copper(I)-HAH1 in the overall functional process.
