Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry.
Metallochaperones are soluble proteins involved in metal transport and regulation in vivo. Copper metallochaperones belong to a structural family of metal binding domains displaying a ferredoxin-like fold (betaalphabetabetaalphabeta) and a consensus metal-binding motif MXCXXC. The metal-binding selectivities for this class of proteins are poorly documented so far. The present study focuses on the measurement of the selectivity of the copper metallochaperone CopZ from Enterococcus hirae for different metal ions using an experimental approach based on electrospray ionization mass spectrometry (ESI-MS). All the metal cations tested, i.e. Cu(I), Cu(II), Hg(II), Cd(II) and Co(II), form specific metal complexes with CopZ. The study of a chemically modified CopZ as well as variants of CopZ in the active site demonstrated that the complexes observed by ESI-MS, i.e. in the gas phase, corresponded to the complexes previously observed by other analytical methods in solution. Competition experiments allowed the classification of the metal ions by increasing affinities for CopZ as follows: Co << Cd < Hg < Cu. A dissociation constant in the range of 20 microM was determined for cobalt. The affinity of CopZ for the other metals tested was found to be higher, with dissociation constants smaller than micromolar.
Urvoas
Agathe
A
CEA, Département d'Ingénierie et d'Etudes des Protéines--Bâtiment 152, C.E. Saclay, F-91191 Gif-sur-Yvette, France.
Amekraz
Badia
B
Moulin
Christophe
C
Le Clainche
Loïc
L
Stöcklin
Reto
R
Moutiez
Mireille
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016983:Enterococcus / Q000378:metabolism*; D008670:Metals / Q000032:analysis / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; C092379:CopZ protein, Enterococcus hirae; D008670:Metals; D018832:Molecular Chaperones; D015534:Trans-Activators