A Key Structural Role for the Active Site Type 3 Copper Ions in Human Ceruloplasmin

Human ceruloplasmin is a copper containing serumglycoprotein with multiple functions. The crystal structureshows that its six domains are arranged in threepairs with a pseudo-ternary axis. Both the holo and apoforms of human ceruloplasmin were studied by size exclusionchromatography and small angle x-ray scatteringin solution. The experimental curve of the holo formdisplays conspicuous differences with the scatteringpattern calculated from the crystal structure. Once thecarbohydrate chains and flexible loops not visible in thecrystal are accounted for, remaining discrepancies suggestthat the central pair of domains may move as awhole with respect to the rest of the molecule. The quasisymmetricalcrystal structure therefore appears to bestabilized by crystal packing forces. Upon copper removal,the scattering pattern of human ceruloplasminexhibits very large differences with that of the holoprotein,which are interpreted in terms of essentially preserveddomains freely moving in solution around flexiblelinkers and exploring an ensemble of openconformations. This model, which is supported by theanalysis of domain interfaces, provides a structural explanationfor the differences in copper reincorporationinto the apoprotein and activity recovery between humanceruloplasmin and two other multicopper oxidases,ascorbate oxidase and laccase. Our results demonstratethat, beyond catalytic activity, the three-copper clusterat the N-terminal-C-terminal interface plays a crucialrole in the structural stability of human ceruloplasmin.[...]