Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character

Abstract

Abstract:After the resolution of the 3D structure of the Cd 9 -aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M 9 -LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M 6 -LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu 4 -aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

Metallothioneins of the urochordateOikopleura dioicahave Cys-rich tandem repeats, large size and cadmium-binding preference

“…48 The selective advantage that MTs with high metal-binding capacity might provide to a marine animal as O. dioica is still unknown, but it has been suggested that the evolution of multi-domain MTs in some animal species (e.g. Littorina littorea and Pomatias elegans with 3-domain MTs [49][50][51] Crassostrea virginica with 4-domain MTs, 52 and Alinda biplicata with 10-domain MTs 53 ) might have improved their response to metal stress and adverse environmental conditions, offering a selective advantage to organisms that experience a greater exposure to metals due to their ecological niche. 50,52…”

Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae)

“…In other snail species, for example, the Cd-binding stoichiometry increases from 6 to 9 Cd 2+ ions bound per protein molecule if an additional metal-binding domain is added to the protein through evolutionary optimization. This was experimentally demonstrated by NMR or mass spectrometry for the Cd-specific MT of Littorina littorea [26,28] (Figure 8). Interestingly, in Cu-specific MTs of fungi such as Tremella mesenterica and Crypotococcus neoformans, too, the addition of Cys-containing tandem repetitions increases their Cu-binding capacity [49,78,85].…”

“…Since the three-domain MT of Littorina littorea clearly exhibits Cd-selective binding features [28] that were confirmed by solution NMR [26], it is suggested that, based on the high degree of sequence similarity, the two md-MTs of A. biplicata might also be Cd-specific. It also appears that both md-MTs are more closely related to other snail CdMTs, including those of Helix pomatia and Cornu aspersum (61-69% identity), than to the respective aminoacid sequence of snail CuMT isoforms (48-54% identity) ( Figure S3B) [19].…”

“…The marine periwinkle Littorina littorea and its terrestrial relative Pomatias elegans, for example, have increased the metal-binding capacity of their Cd-selective MTs by addition of a third Cd-binding domain, derived from a duplication of their N-terminal domain [26,27]. Consequently, these newly discovered three-domain MTs have increased their binding stoichiometry from 6 to 9 Cd 2+ ions [26,28]. Our most recent studies indicate the presence of multi-domain MTs (md-MTs) with as much as eight (8) metal-binding domains (8md-MT) in an MT of the freshwater snail Marisa cornuarietis, and with nine (9) or even ten (10) metal-binding domains (9md-MT and 10md-MT) in two MT isoforms of the terrestrial snail A. biplicata [19].…”

“…Alignment of putative multi-domain MT proteins from A. biplicata with the previously published 3md-MT from Littorina littorea[26,28]. N-terminal domain sequences (N1-N9) are highlighted in green, whereas the C-terminal domains are colored in blue.…”