Evolution of metallothioneins in gastropods

In this project, we aim to track and understand the evolutionary pathways of gastropod Metallothioneins (MTs) from metal-unspecific proteins towards metal-specific MT isoforms via structural and functional differentiation. In particular, we are interested in understanding the structural adaptations through which this specificity has been achieved, on which time scale these have happened, and how these structure modifications have been translated into distinct metal-related protein functions. To achieve our goals, we intend to apply a multi-disciplinary and -methodical approach: First, we will use a phylogenetic strategy to track the evolutionary path and to comprehend key events during development of metal- specificity in MTs of selected species from different gastropod taxa. Second, we want to apply mutagenesis and protein bioengineering to construct MT chimeras and mutation variants with only a small preference of one metal over the other in order to spot modifications critical for metal selectivity. Third, we want to determine, by methods of NMR, the tertiary structure of metal-specific native and experimentally mutated snail MTs with the aim to better understand which primary structure modifications are required to alter the protein folding such that different metal ions with differential stoichiometric and coordination requirements are bound preferentially. And fourth, we want to study the functional implications of such structural diversifications in gastropod MTs in terms of their metal- related physiological functions. The project will be realized via a series of cooperations. The main cooperation (as a DACH project) will be established between Dr. Dallinger of the University of Innsbruck (Institute of Zoology) and Dr. Oliver Zerbe from the University of Zürich (Institute of Organic Chemistry). Dr. Dallinger will be responsible for coordination and contribute to the project by adding biochemical and physiological know-how, whereas Dr. Zerbe will focus on the structural characterization of selected native and mutated MT variants by solution NMR. Dr. Lindner of the Innsbruck Medical University (Division of Clinical Biochemistry) will support the project as a national cooperation partner with advanced protein-analytical expertise. Absolutely essential for the success of the project will be, moreover, the cooperations with Dr. Silvia Atrian of the University of Barcelona (Department of Genetics) and Dr. Mercé Capdevila of the Autonomous University of Barcelona (Department of Chemistry). Whereas Dr. Atrian will be responsible for the systematic recombinant production of selected MT proteins and mutation variants, Dr. Capdevila will take over the produced samples and screen their metal-binding features (homogeneity, complex stability, metal stoichiometry, etc.) by spectrophotometric methods and MS spectrometry. Based on this preliminary screening, samples will then either be discarded or considered, due to their emerging interesting features, for more detailed biochemical, structural and physiological studies by all other project partners.

With about 80 000 extant species, modern snails (Gastropoda) represent one of the most important classes of mollusks (Mollusca), whose origin can be traced back to the Cambrian era, about 500 million years ago. Due to their exceptional capacity to cope with adverse environmental conditions, snails were able to adapt to nearly all realms of the earth. Several gastropod species can accumulate and detoxify high concentrations of metals such as cadmium and lead in their tissues. Consequently, metal accumulating snail species have repeatedly been used for biomonitoring of environmental metal pollution. Their cadmium detoxifying capacity is based on complexation of toxic metals by metallothioneins (MTs), a family of ubiquitous detoxifying proteins so far known from all animal species. In contrast to most other animals, many snail MTs possess a highly selective binding preference for cadmium, conferring to the respective snail species an exceptional cadmium detoxifying efficiency. Evidently, this capacity must have evolved during evolution long ago, prior to the onset of increasing cadmium emissions due to anthropogenic pollution. In the present project, we wanted to test this hypothesis by exploring the evolutionary origin of cadmium- selective gastropod MTs, the reasons why they have been invented, and how they were subjected to metal-specific differentiation to the present day. In an interdisciplinary cooperation with colleagues from Spain and Switzerland, we applied methods of molecular, biochemical and structural biology to resolve the three-dimensional structure of cadmium- specific MTs from two different species, the terrestrial Roman snail and the littoral periwinkle. By applying a combined approach of zoology and evolutionary biology, we learned that cadmium-specific MTs were invented 430 million years ago in different gastropod clades through convergent evolution of diverging metal-binding domains, accompanied by structural optimization of their cadmium-binding specificity. Overall, this evolutionary process was driven by increased cadmium emissions through repeated super-volcanic activities through the earths history. Since the onset of the cretaceous era, about 145 million years ago, snail MTs have diversified into various kinds or even lost metal specificity through gastropod adaptation to novel habitats with different cadmium background concentrations. This demonstrates that cadmium although being highly toxic and lacking essential biological functions has been a principal evolutionary driver in shaping one of the most important metal detoxifying protein families.