Engineering of metallothionein-3 neuroinhibitory activity into the inactive isoform metallothionein-1

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimer's disease, exhibits neuroinhibitory activity in vitro and a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer (113)Cd NMR experiments performed on the (113)Cd-reconstituted wild-type and mutant Cd(7)-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd(3)-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the (5)TCPCP(9) motif. The functional significance of such heterogeneous cluster ensemble is discussed.