title: A key structural role for active site type 3 copper ions in human ceruloplasmin.
abstract: Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.
authors.affiliation: LURE, Bat. 209d, Université Paris-Sud, B.P. 34, F91898 Orsay Cedex, France. patrice.vachette@lure.u-psud.fr
authors.forename: Patrice
authors.initials: P
authors.lastname: Vachette
authors.forename: Enrico
authors.initials: E
authors.lastname: Dainese
authors.forename: Vadim B
authors.initials: VB
authors.lastname: Vasyliev
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Muro
authors.forename: Mariano
authors.initials: M
authors.lastname: Beltramini
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Filippis
authors.forename: Benedetto
authors.initials: B
authors.lastname: Salvato
mesh_terms: D001665:Binding Sites; D002240:Carbohydrate Sequence; D002570:Ceruloplasmin / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012542:Scattering, Radiation
chemical_list: D003300:Copper; D002570:Ceruloplasmin