Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3

The brain specific member of the metallothionein (MT) family of proteins, metallothionein-3, inhibits the growth and survival of neurons, in contrast to the ubiquitous mammalian MT isoforms, MT-1 and MT-2, that are found in most tissues and are thought to function in metal ion homeostasis and detoxification. Solution NMR was utilized to determine the structural and dynamic differences of MT-3 from MT-1 and 2. The high-resolution solution structure of the C-terminal alpha-domain of recombinant mouse MT-3 revealed a tertiary fold very similar to MT-1 and 2, except for a loop that accommodates an acidic insertion relative to these isoforms. This loop was distinguished from the rest of the domain by dynamics of the backbone on the nano- to picosecond time-scale shown by (15)N relaxation studies and was identified as a possible interaction site with other proteins. The N-terminal beta-domain contains the region responsible for the growth inhibitory activity, a CPCP tetrapeptide close to the N-terminus. Because of exchange broadening of a large number of the NMR signals from this domain, homology modeling was utilized to calculate models for the beta-domain and suggested that while the backbone fold of the MT-3 beta-domain is identical to MT-1 and 2, the second proline responsible for the activity, Pro9, may show structural heterogeneity. (15)N relaxation analyses implied fast internal motions for the beta-domain. On the basis of these observations, we conclude that the growth inhibitory activity exhibited by MT-3 is a result of a combination of local structural differences and global dynamics in the beta-domain.