Histidines promote zinc over cadmium binding to the single type 4 metallothionein from Great Millet (Sorghum bicolor)
Agnieszka Mierek-Adamska, Jose Gutierrez-Marcos, Claudia A. Blindauer
Zinc and cadmium share similar chemical properties; however, while zinc is an indispensable microelement involved in several physiological processes, cadmium is highly toxic. Cadmium toxicity results at least to some extent from replacing zinc (and other metals) from their active sites in enzymes and other proteins. This highlights why the correct population of metalloproteins with metals is crucial for proper cellular metabolism. In the face of growing demand for food, both in terms of quantity and quality, a rapid development of crop cultivars containing a higher amount of bioavailable zinc in the edible parts of plants, crucially without the simultaneous accumulation of cadmium, is imperative. Type 4 plant metallothioneins (pMT4s) are seed-specific proteins for which a potential role as a zinc specificity filter has been proposed. It was suggested that two conserved histidine residues are key for discrimination between zinc and cadmium. In this study, we analysed the metal-binding properties of Sorghum bicolor pMT4 (SbMT4) wild-type and mutant proteins with histidine/s replaced by tyrosine/s (H32Y, H40Y, and H32Y/H40Y) using mass spectrometry, elemental analysis, and NMR spectroscopy. SbMT4 is a Zn-thionein, but unexpectedly, it was also fully folded in the presence of cadmium – owing to a zinc ion remaining in the mononuclear Cys2His2 site in domain II. All three mutant proteins were misfolded in the presence of either zinc or cadmium, but increased Cd-to-protein stoichiometry was observed. The presence of histidines impacted SbMT4 metal selectivity when expressed in bacterial cells, but did not affect Zn/Cd accumulation in transgenic Arabidopsis thaliana plants.