The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Abstract

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.

Metal-binding properties and structural characterization of a self-assembled coiled coil: Formation of a polynuclear Cd–thiolate cluster

“…The resulting isosbestic points appearing at 232 and 260 nm indicate a step-wise formation of two different metallated products during the course of the cadmium titration at pH 7. It is noted that a similar bathochromic shift of 6 nm for the S − →Cd 2+ LMCT band was previously observed in the cadmium reconstitution of mammalian metallothionein-1 [66] and crustacean metallothionein-2 [67]. Such shifts were assigned to the deconvolution of the difference absorption spectrum of the AQ-C16C19 transition of a mononuclear tetrahedral complex of Cd 2+ with four terminal thiolates to a Cd-thiolate polynuclear site featuring bridging thiolate ligands [66].…”

Metallothioneins, Unconventional Proteins from Unconventional Animals: A Long Journey from Nematodes to Mammals

“…This MT-2 is 64 amino acids long, contains 21 cysteines and is characterized by two Cys-Cys-Cys triplets, each domain containing one triplet. In terms of MT-2’s metal-binding capacity, the fully Zn/Cd metallated thionein could present up to eight metal ions and up to 12 for Cu(I) [36]. Crustaceans present a particular Cu(I) oxygen carrier protein, haemocyanin, and should be characterized by specific homeostatic mechanisms.…”

Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis

“…In Metazoans, MTs with one or more Cys-Cys-Cys triplets have been found in oligochaetes, mollusks and crustaceans [27][28][29][30][31]. Showing ability for preferentially binding Cu ions [32], these atypical forms of MT in mollusks and crustaceans have been associated with the presence of hemocyanin for oxygen transport, suggesting that these atypical MTs are involved in copper homeostasis associated with the synthesis and degradation of hemocyanin.…”

“…(in A, three biological replicates each consisting of three pooled tadpoles; in B, n = 3). three-metal or the four-metal thiolate cluster [31]. Circular dichroism and UV spectra performed on the b-domain of the human metallothionein 1b demonstrated that the additional cysteine positioned to form the cysteine triplet characterizing this isoform did not perturb the overall structure of the protein or the formation of the metalthiolate clusters [35].…”