Structure and Biology of Multi-Domain Metallothioneins

Gastropoda (snails) represent an ancient animal clade known since the cambrian era, characterized by their high stress resistance and exceptional detoxification capacity for highly toxic heavy metals such as cadmium (Cd). This capability is based on the specific binding and inactivation of Cd by the so-called metallothioneins (MTs). These are mostly low-molecular weight, heavy metal-binding proteins with typically two to three structural subunits (domains), each of them possessing nine sulfur atoms, through which three divalent Cd ions can be bound. Recent studies have shown that modern gastropod species, in particular, possess higher - molecular weight MTs with up to eleven Cd-binding domains, called multi-domain MTs (md-MTs), whose detoxification capacity for toxic Cd ions is, therefore, manifold enhanced. The expression and synthesis of these proteins can be induced on a short-term scale by the presence of environmental Cd, which further expedites the process of detoxification. In all so-far known two or three-domain gastropod MTs, the metal- binding domain situated at the C-terminal part of the protein (called Beta 2-domain) differs significantly from the remaining so-called Beta 1-domains due to its capacity for initiating the loading of Cd to the whole protein, thereby intensifying the metal binding strength. It is not clear, however, if this does also hold for gastropod md-MTs, and which molecular mechanisms may be responsible for the initiating function of the Beta 2-domain upon Cd binding. In the present research project, we want to synthesize some known and novel md-MTs from diverse gastropod species by recombinant expression, either in their native structure or after targeted structural alteration by interchanging the positions of Beta 2 and Beta 1-domains, in order to examine if these chimeric mutations may have an impact on their biological function. To this aim, we will transform native and chimeric md-MTs in cell cultures and living nematodes (Caenorhabditis elegans) and assess their detoxification capacity in the respective biological systems. Moreover, the metal binding capacity and the three-dimensional structure of these md-MTs will be elucidated by means of mass spectrometry and NMR (Nuclear Magnetic Resonance Spectrometry). We expect from our studies to gain extensive knowledge of the molecular mechanisms of Cd detoxification and hope to provide a better understanding of the relationships between the structure of these proteins and their detoxification capacity. The studies will be directed and coordinated by Dr. Reinhard Dallinger und Dr. Veronika Pedrini-Martha from the department of zoology of the University of Innsbruck. This will happen in close cooperation with our collaboration partners in Innsbruck (Dr. Pidder Jansen-Dürr, department for Biomedical Aging Research), at the University of Zürich, Switzerland (Dr. Oliver Zerbe, Department of Biochemistry), und at two university departments in Barcelona, Spain (Dr. Mercé Capdevila, Department of Chemistry of the Autonomous University of Barcelona and Dr. Ricard Albalat, Department of Genetics, University of Barcelona).

Metallothioneins (MTs) are metal-binding proteins present in all animal groups. They are responsible for the detoxification and homeostatic regulation of important physiological metals, including copper (Cu), zinc (Zn) and cadmium (Cd). As demonstrated previously, numerous gastropod species (snails) of the phylum Mollusca possess Cd-specific MTs that have evolved through evolution. Our recent research has revealed that some of these MTs have a multi-domain structure, in which one or more metal-binding domains have multiplied in a repetitive manner. This has led to the development of MTs with up to 10 metal-binding domains, as discovered recently in the terrestrial clausiliid snail Alinda biplicata (A.b.). These proteins are therefore called multi-domain metallothioneins (md-MTs). This project focused on the structure and metal-binding properties of md-MTs, as well as their evolutionary and ecophysiological significance, in species of gastropods (snails) from terrestrial, aquatic and marine habitats. As a first step, the frequency and genetic variability of md-MTs in gastropods was investigated. In some species, moreover, the physiological function of md-MTs after metal and stress exposure was examined using an in vivo approach. Based on this, some of these md-MTs and their mutant variants with altered domain sequences and reduced domain numbers were recombinantly expressed to characterise their structure and metal-binding properties using spectrophotometric methods, mass spectrometry and NMR. md-MT constructs from A.b. were then transfected or expressed in three different eukaryotic cell systems: yeast cells, mouse fibroblasts and the nematode worm Caenorhabditis elegans (C.e.). The aim was to verify whether, and to what extent, the md-MTs of A. b. (and their mutants) expressed in these systems actually resulted in increased metal resistance. The most important findings can be summarised as follows. 1. MD-MTs are widespread among gastropods. They have been detected in terrestrial, marine and freshwater species. 2. Throughout, the species-specific md-MTs exhibited high Cd-selectivity in their metal binding and served primarily for Cd detoxification. 3. The expression of native and mutated md-MTs from A.b. led to increased Cd resistance in all eukaryotic cell systems and in C.e. after appropriate exposure, and even (in C. e.) to increased physiological fitness compared to the wild-type strain. 4. NMR was used to elucidate the 3D structure and dynamics of metal loading on md-MTs from A.b. and a deep-sea snail (Chrysomallon squamiferum) (C.s.). This revealed that md-MTs do not have a globular structure, but consist of a string of metal-binding domains ('beads on string'). 5. High-pressure NMR spectroscopy finally demonstrated that the 3D structure of md-MTs from deep-sea snails (C. s.) can adapt to the high-pressure conditions of their habitat by incorporating water molecules, thereby maintaining flexibility, which is essential for metal binding.