National Synchrotron Light Source Seminar

Metallothioneins (MTs) are low molecular weight proteins with high binding capacity for essential metals including Zn and Cu but they also tenaciously bind toxic metals such as Cd and Hg. Metal ions in the protein are coordinated by the cysteine sequence motifs (cys-motifs) which have been used as a basis for classification of MTs as a superfamily. Recent studies indicate that all members of this family may not have a single unifying function; some MTs may participate in metal homeostasis, others may play a role in heavy metal detoxification and some others may be involved in metal ion transport. Lack of direct biochemical and biophysical data on purified proteins hinders establishment of sequence-structure-metal-binding and function relationships in MTs from different sources.
We identified an MT from a Cd tolerant strain of Triticum durum wheat and cloned and expressed this protein (dMT) in E. coli for structural investigations. dMT was shown to be a type I plant MT with two terminal metal-binding clusters separated by a hinge region significantly longer than that observed in its mammalian counterparts (40 residues vs. 2-8 residues). Metal to protein ratio was found to be 5±1 Cd/dMT molecule. dMT structure was predicted by homology modeling and ab initio calculations which yielded a dumbbell shape with Cd-binding clusters at N- and C-termini separated by the hinge region with secondary structure elements. In parallel small angle X-ray solution scattering (SAXS) data form dMT and its mutants were used for modeling the molecular shape by ab initio calculations and simulated annealing methods. Results confirmed the extended shape and the structural parameters were found to be consistent with a folded hinge region. Monitoring Cd-binding and release by circular dichroism indicated non-cooperative metal-binding and that dMT retained a residual fold even in the apo-form. These findings point to a functional role where dMT may be involved in metal transport. A folded