Abstract
The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 A, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis.
Highlights
Human ceruloplasmin, human ceruloplasmin (hCP), is a member of the multi-copper oxidase family of enzymes that includes ascorbate oxidase and the laccases
The first X-ray structural study of human ceruloplasmin was reported in 1996 (Zaitseva et al, 1996) and confirmed that the molecule is comprised of six cupredoxintype domains arranged in a triangular array
Each of the mononuclear copper ions is coordinated to a cysteine and two histidine residues and those in domains 4 and 6 coordinate weakly to a methionine residue; in domain 2, the methionine is replaced by a leucine residue at a van der Waals contact distance from the cation
Summary
HCP, is a member of the multi-copper oxidase family of enzymes that includes ascorbate oxidase and the laccases. The size of these crystals still needs to be improved, but is has been possible to collect X-ray data to 2.8 Ausing the European Synchrotron Radiation Facility at Grenoble, France This modest increase in resolution, together with the cryotemperature, which minimizes the thermal motion of the atoms in the crystal, has enabled further X-ray studies resulting in new structural information on the hCP molecule, including the identification of Ca2+-binding and Na+-binding sites; these findings are reported here. A ferroxidase role of the enzyme in plasma appears to have been substantiated and in this context ceruloplasmin may serve to assist the release of iron from cells prior to uptake of the metal by the transferrin (Frieden & Hsieh, 1976; Lindley et al, 1999) Another series of structural studies (Zaitsev et al, 1999) located the binding sites for an inhibitor, azide, and various amine substrates.
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