Engineering of Metallothionein-3 Neuroinhibitory Activity into the Inactive Isoform Metallothionein-1

Núria Romero-Isart, Laran T. Jensen, Oliver Zerbe, Dennis R. Winge, Milan Vašák
2002 Journal of Biological Chemistry  
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
more » ... e 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. This paper is available on line at http://www.jbc.org 37023 by guest on July 25, 2018 http://www.jbc.org/ Downloaded from 113 Cd resonances originating from the 3-metal cluster (Fig. 3C). Despite the large thermodynamic stability of the metal-thiolate bonds in MTs, they are kinetically very labile. The occur-FIG. 3. 133-MHz 113 Cd NMR spectra of mouse 113 Cd 7 -MT-1 (A), 113 Cd 7 -S6P,S8P MT-1 (B), and 113 Cd 7 -S8P,S8P؉T5 MT-1 (C) at 298 and 323 K. 20-Hz line broadening was applied during processing. The arrows indicate the reversibility of the process. The small signals (E) are irreversible and originate from minor sample degradation during the measurement at 323 K. Engineering Neuronal Bioactivity into
doi:10.1074/jbc.m205730200 pmid:12130647 fatcat:y3lgiqyfozdj3lo2w3apajpv6y