Plant metallothionein domains: functional insight into physiological metal binding and protein folding

被引:64
作者
Domenech, Jordi
Mir, Gisela
Huguet, Gemma
Capdevila, Merce
Molinas, Marisa
Atrian, Silvia
机构
[1] Univ Barcelona, Fac Biol, Dept Genet, E-08028 Barcelona, Spain
[2] Univ Girona, Dept Biol, Girona 17071, Spain
[3] Univ Autonoma Barcelona, Fac Ciencies, Dept Quim, E-08193 Barcelona, Spain
关键词
metallothionein; Quercus suber; separate Cys-rich domains; spacer region; Zn-aggregates; Cu-aggregates; MT dimers; yeast complementation;
D O I
10.1016/j.biochi.2005.11.002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Plant metallothioneins (MTs) differ from animal MTs by a peculiar sequence organization consisting of two short cysteine-rich terminal domains linked by a long cysteine-devoid spacer. The role of the plant NIT domains in the protein structure and functionality is largely unknown. Here, we investigate the separate domain contribution to the in vivo binding of Zn and Cu and to confer metal tolerance to CUP1-null yeast cells of a plant type 2 NIT (QsMT). For this purpose, we obtained three recombinant peptides that, respectively, correspond to the single N-terminal (N25) and C-terminal (C18) cysteine-rich domains of QsMT, and a chimera in which the spacer is replaced with a four-glycine bridge (N25-C18). The metal-peptide preparations recovered from Zn- or Cu-enriched cultures were characterized by ESI-MS, ICP-OES and CD and UV-vis spectroscopy and data compared to full length QsMT. Results are consistent with QsMT giving rise to homometallic Zn- or Cu-MT complexes according to a hairpin model in which the two Cys-rich domains interact to form a cluster. In this model the spacer region does not contribute to the metal coordination. However, our data from Zn-QsMT (but not from Cu-QsMT) support a fold of the spacer involving some interaction with the metal core. On the other hand, results from functional complementation assays in endogenous MT-defective yeast cells suggest that the spacer region may play a role in Cu-QsMT stability or subcellular localization. As a whole, our results provide the first insight into the structure/function relationship of plant MTs using the analysis of the separate domain abilities to bind physiological metals. (c) 2005 Elsevier SAS. All rights reserved.
引用
收藏
页码:583 / 593
页数:11
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