title: Single domain metallothioneins: supermetalation of human MT 1a.
abstract: Metallothioneins are a family of small, cysteine rich proteins that have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as metallochaperones involved in the homeostasis of both essential zinc and copper. We report that human metallothionein 1a, well-known to coordinate 7 Zn(2+) or Cd(2+) ions with 20 cysteinyl thiols, will bind 8 structurally significant Cd(2+) ions, leading to the formation of the supermetalated Cd(8)-βα-rhMT 1a species, for which the structure is a novel single domain. ESI-mass spectrometry was used to determine the exact metalation status of the βα-rhMT. The derivative-shaped CD envelope of Cd(7)-βα-rhMT [peak extrema (+) 260 and (-) 239 nm] changed drastically upon formation of the Cd(8)-βα-rhMT with the appearance of a sharp monophasic CD band centered on 252 nm, a feature indicative of the loss of cluster symmetry. The structural significance of the eighth Cd(2+) ion was determined from a combination of direct and indirect (113)Cd nuclear magnetic resonance (NMR) spectra. In the case of Cd(8)-βα-rhMT, only four peaks were observed in the direct (113)Cd NMR spectrum. Significantly, while both of the isolated domains can be supermetalated forming Cd(4)-β-rhMT and Cd(5)-α-rhMT, Cd(8)-βα-rhMT and not Cd(9)-βα-rhMT was observed following addition of excess Cd(2+). We propose that both domains act in concert to coordinate the eighth Cd(2+) atom, and furthermore that this interaction results in a coalescence of the two domains leading to collapse of the two-domain structure. This is the first report of a possible single-"superdomain" metallothionein structure for Zn(2+) and Cd(2+) binding mammalian proteins. A computational model of a possible single-domain structure of Cd(8)-βα-rhMT is described.
authors.affiliation: Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada.
authors.forename: Duncan E K
authors.initials: DE
authors.lastname: Sutherland
authors.forename: Mathew J
authors.initials: MJ
authors.lastname: Willans
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D002104:Cadmium / Q000737:chemistry; D002942:Circular Dichroism / Q000379:methods; D006706:Homeostasis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008668:Metallothionein / Q000737:chemistry*; D008968:Molecular Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013696:Temperature; D013917:Thrombin / Q000737:chemistry; D014466:Ultraviolet Rays; D015032:Zinc / Q000737:chemistry
chemical_list: C530099:MT1A protein, human; D011506:Proteins; D002104:Cadmium; D008668:Metallothionein; D013917:Thrombin; D015032:Zinc