Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity.
Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2 approximately four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form alpha-helical secondary structure in TFE-water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% alpha-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.
Palumaa
Peep
P
Department of Gene Technology, Tallinn Technical University Akadeemia tee 23, 12618 Tallinn, Estonia. peepp@staff.ttu.ee
Tammiste
Indrek
I
Kruusel
Keiu
K
Kangur
Liina
L
Jörnvall
Hans
H
Sillard
Rannar
R
D000544:Alzheimer Disease / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D002942:Circular Dichroism; D019610:Cytoprotection; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D000090023:Metallothionein 3; D008670:Metals / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017382:Reactive Oxygen Species / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity
D006131:Growth Inhibitors; D000090023:Metallothionein 3; D008670:Metals; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D002104:Cadmium; D008668:Metallothionein; D015032:Zinc