The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets.
Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M(8) (II)-MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn(6)-MTC and Cd(7)-MTC species from M(8) (II)-MTC after treatment with Chelex 100. The NMR characterization of Cd(7)-MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal-Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1-31) reveal a Cd(3)Cys(9) cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32-64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu(8-9)-MTC). The subsequent generation of Cu(12)-MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.
Serra-Batiste
Montserrat
M
Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Cols
Neus
N
Alcaraz
Luis A
LA
Donaire
Antonio
A
González-Duarte
Pilar
P
Vasák
Milan
M
D000818:Animals; D001665:Binding Sites; D003386:Brachyura / Q000737:chemistry*; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D008670:Metals; D011994:Recombinant Proteins; D002104:Cadmium; D003300:Copper; D008668:Metallothionein; D015032:Zinc; D003545:Cysteine