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).