Discrete roles of copper ions in chemical unfolding of human ceruloplasmin.
Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six beta-barrel domains with one type 1 copper in each of domains 2, 4, and 6; the remaining copper ions form a catalytic trinuclear cluster, one type 2 and two type 3 coppers, at the interface between domains 1 and 6. We have characterized urea-induced unfolding of holo- and apo-forms of CP by far-UV circular dichroism, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid binding, visible absorption, copper content, and oxidase activity probes (pH 7, 23 degrees C). We find that holo-CP unfolds in a complex reaction with at least one intermediate. The formation of the intermediate correlates with decreased secondary structure, exposure of aromatics, loss of two coppers, and reduced oxidase activity; this step is reversible, indicating that the trinuclear cluster remains intact. Further additions of urea trigger complete protein unfolding and loss of all coppers. Attempts to refold this species result in an inactive apoprotein with molten-globule characteristics. The apo-form of CP also unfolds in a multistep reaction, albeit the intermediate appears at a slightly lower urea concentration. Again, correct refolding is possible from the intermediate but not the unfolded state. Our study demonstrates that in vitro equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages. When the catalytic site is finally destroyed, refolding is not possible at neutral pH. This implies a mechanistic role for the trinuclear metal cluster as a nucleation point, aligning domains 1 and 6, during CP folding in vivo.
Sedlak
Erik
E
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, Texas, USA.
Wittung-Stafshede
Pernilla
P
D002412:Cations / Q000737:chemistry; D002570:Ceruloplasmin / Q000737:chemistry*; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014508:Urea / Q000737:chemistry
D002412:Cations; D020035:Holoenzymes; D003300:Copper; D014508:Urea; D002570:Ceruloplasmin