Abstract:Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs… Show more
“…Evolutionarily, MTs are observed to undergo reshuffling, rearrangement, multiplication or formation of de novo domains, which provides new functional abilities, such as increasing the frequency of metal ion binding or increasing the affinity for a specific metal ion. 98 In this way LlMT increases the buffering capacity and range of buffered pZn compared to two-domain MTs. Moreover, such an evolutionary procedure in the form of increasing the size of a given protein is more energetically advantageous than gene duplication, as was proven during research on yeast.…”
Metallothioneins (MTs) are small, Cys-rich proteins present in various but not all organisms, from bacteria to humans. They participate in zinc and copper metabolism, toxic metals detoxification and protection against reactive species. Structurally, they contain one or multiple domains, capable of binding a variable number of metal ions. For experimental convenience, biochemical characterization of MTs is mainly performed on Cd(II)-loaded proteins, frequently omitting or limiting Zn(II) binding features and related functions. Here, by choosing ten MTs with relatively well-characterized structures from animals, plants, and bacteria, we focused on poorly investigated Zn(II)-to-protein affinities, stability-structure relations, and the speciation of individual complexes. For that purpose, MTs were characterized in terms of stoichiometry, pH-dependent Zn(II) binding, and competition with chromogenic and fluorescent probes. To shed more light on protein folding and its relation with Zn(II) affinity, reactivity of variously Zn(II)-loaded MTs was studied by DTNB oxidation in the presence of mild chelators. The results show that animal and plant MTs, despite their architectural differences, demonstrate the same affinities to Zn(II), varying from nano- to low picomolar range. Bacterial MTs bind Zn(II) more tightly but, importantly, with different affinities from low picomolar to low femtomolar range. The presence of weak, moderate and tight zinc sites is related to the folding mechanisms and internal electrostatic interactions. Differentiated affinities of all MTs define their zinc buffering capacity required for Zn(II) donation and acceptance at various free Zn(II) concentrations (pZn levels). The data demonstrate critical roles of individual Zn(II)-depleted MT species in zinc buffering processes.
“…Evolutionarily, MTs are observed to undergo reshuffling, rearrangement, multiplication or formation of de novo domains, which provides new functional abilities, such as increasing the frequency of metal ion binding or increasing the affinity for a specific metal ion. 98 In this way LlMT increases the buffering capacity and range of buffered pZn compared to two-domain MTs. Moreover, such an evolutionary procedure in the form of increasing the size of a given protein is more energetically advantageous than gene duplication, as was proven during research on yeast.…”
Metallothioneins (MTs) are small, Cys-rich proteins present in various but not all organisms, from bacteria to humans. They participate in zinc and copper metabolism, toxic metals detoxification and protection against reactive species. Structurally, they contain one or multiple domains, capable of binding a variable number of metal ions. For experimental convenience, biochemical characterization of MTs is mainly performed on Cd(II)-loaded proteins, frequently omitting or limiting Zn(II) binding features and related functions. Here, by choosing ten MTs with relatively well-characterized structures from animals, plants, and bacteria, we focused on poorly investigated Zn(II)-to-protein affinities, stability-structure relations, and the speciation of individual complexes. For that purpose, MTs were characterized in terms of stoichiometry, pH-dependent Zn(II) binding, and competition with chromogenic and fluorescent probes. To shed more light on protein folding and its relation with Zn(II) affinity, reactivity of variously Zn(II)-loaded MTs was studied by DTNB oxidation in the presence of mild chelators. The results show that animal and plant MTs, despite their architectural differences, demonstrate the same affinities to Zn(II), varying from nano- to low picomolar range. Bacterial MTs bind Zn(II) more tightly but, importantly, with different affinities from low picomolar to low femtomolar range. The presence of weak, moderate and tight zinc sites is related to the folding mechanisms and internal electrostatic interactions. Differentiated affinities of all MTs define their zinc buffering capacity required for Zn(II) donation and acceptance at various free Zn(II) concentrations (pZn levels). The data demonstrate critical roles of individual Zn(II)-depleted MT species in zinc buffering processes.
“…The two-domain organization is overall conserved among gastropod MTs, even though exceptions can be found. 19 , 39 The advantage of this two-domain organization is, however, less clear since both domains can act in isolation despite forming contacts in the native proteins. 20 , 39 Further, the degree to which either domain is conserved varies, with C-domains being generally more conserved among gastropod MTs.…”
Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn2+, Cd2+, and Cu+, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use NMR to follow the evolution of Cd2+ and Cu+ binding from the reconstructed ancestral Stylommatophora MT to the three Helix pomatia MT (HpMT) isoforms. Information obtained from [15 N,1H, ]-HSQC spectra and T2 relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd2+ complexes and closely resembles the Cd2+-specific HpCdMT isoform, suggesting a role in Cd2+ detoxification for the ancestral protein. All Cu+-MT complexes, including the Cu+-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu+-specific HpCuMT isoforms form Cu+ complexes with comparable characteristics. It is possible to follow how Cd2+ and Cu+ binding changed throughout evolution. Interestingly, Cu+ binding improved independently in the lineages leading to the unspecific and the Cu+-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.
“…This has led to a multiplication of the binding stoichiometry for Cd 2+ (or other divalent metal ions) in the corresponding MTs (Pedrini-Martha et al 2020 ). The capacity to form multidomain MTs seems to be an ancient character of molluscs (Jenny et al 2004 , 2016 ; Nam and Kim 2017 ; Calatayud et al 2022 ) and has also been observed in the class of bivalves and in other animal phyla, too (Calatayud et al 2018 ). Fig.…”
Section: The Multifarious World Of Gastropod Mtsmentioning
This is a critical review of what we know so far about the evolution of metallothioneins (MTs) in Gastropoda (snails, whelks, limpets and slugs), an important class of molluscs with over 90,000 known species. Particular attention will be paid to the evolution of snail MTs in relation to the role of some metallic trace elements (cadmium, zinc and copper) and their interaction with MTs, also compared to MTs from other animal phyla. The article also highlights the important distinction, yet close relationship, between the structural and metal-selective binding properties of gastropod MTs and their physiological functionality in the living organism. It appears that in the course of the evolution of Gastropoda, the trace metal cadmium (Cd) must have played an essential role in the development of Cd-selective MT variants. It is shown how the structures and Cd-selective binding properties in the basal gastropod clades have evolved by testing and optimizing different combinations of ancestral and novel MT domains, and how some of these domains have become established in modern and recent gastropod clades. In this context, the question of how adaptation to new habitats and lifestyles has affected the original MT traits in different gastropod lineages will also be addressed. The 3D structures and their metal binding preferences will be highlighted exemplarily in MTs of modern littorinid and helicid snails. Finally, the importance of the different metal requirements and pathways in snail tissues and cells for the shaping and functionality of the respective MT isoforms will be shown.
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