Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae.
We studied the interaction of several metal ions with the copper chaperone from Enterococcus hirae (EhCopZ). We show that the stoichiometry of the protein-metal complex varies with the experimental conditions used. At high concentration of the protein in a noncoordinating buffer, a dimer, (EhCopZ)2-metal, was formed. The presence of a potentially coordinating molecule L in the solution leads to the formation of a monomeric ternary complex, EhCopZ-Cu-L, where L can be a buffer or a coordinating molecule (glutathione, tris(2-carboxyethyl)phosphine). This was demonstrated in the presence of glutathione by electrospray ionization MS. The presence of a tyrosine close to the metal-binding site allowed us to follow the binding of cadmium to EhCopZ by fluorescence spectroscopy and to determine the corresponding dissociation constant (Kd = 30 nm). Competition experiments were performed with mercury, copper and cobalt, and the corresponding dissociation constants were calculated. A high preference for copper was found, with an upper limit for the dissociation constant of 10-12 m. These results confirm the capacity of EhCopZ to bind copper at very low concentrations in living cells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ.
Département d'Ingénierie et d'Etudes des Protéines, Direction des Sciences du Vivant, CEA Saclay, Gif sur Yvette, France.
Agathe
A
Urvoas
Mireille
M
Moutiez
Clément
C
Estienne
Joël
J
Couprie
Elisabeth
E
Mintz
Loïc
L
Le Clainche
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C092379:CopZ protein, Enterococcus hirae; D008670:Metals; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D015534:Trans-Activators; D003300:Copper