Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

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.

Figure 1

The overall organization of the ceruloplasmin molecule, showing the six cupredoxin domains (domains 1, 2, 3, 4, 5 and 6 in red, orange, yellow, green, blue and purple, respectively) and the locations of the metal-binding sites: Cu²⁺ as blue spheres, Ca²⁺ as an olive-green sphere and Na⁺ as red spheres. The relatively planar bottom surface and the protuberances at the top surface are clearly visible. The figures were prepared with the PyMOL program (DeLano, 2002 ▶).

Figure 2

The trinuclear cluster between domains 1 and 6, showing the water molecule attached to the type 2 copper Cu34. An OMIT electron-density map, F _o − F _c, contoured at 7 r.m.s. clearly confirms the presence of a diatomic species, assumed to be dioxygen, within the type 3 cluster.

Figure 3

The Ca²⁺-binding site in domain 1. (a) Overall view of domain 1, showing the locations of the trinuclear copper cluster and the glycan chain at Asn119. (b) Details of the molecular geometry at the Ca²⁺ centre.

Figure 4

The Na⁺-binding site between domains 5 and 6. (a) An overall view with the main chains of domains 5 and 6 coloured blue and green, respectively. (b) A detailed view of the bonding in the vicinity of the Na4 cation site. In (b) Ile756 is not shown for clarity, although its main-chain N atom forms a hydrogen bond with W115 on the left-hand side of the figure (see Table 6 ▶).