The cadmium binding domains in the metallothionein isoform Cd(7)-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy.
The metal-thiolate connectivity of recombinant Cd(7)-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of (15)N T (1) and T (2) relaxation times and steady state {(1)H}-(15)N heteronuclear NOEs. The (113)Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of (113)Cd NMR signals. The complete assignment of cysteine Halpha and Hbeta proton resonances and the analysis of 2D (113)Cd-(113)Cd COSY and (1)H-(113)Cd HMQC type spectra allowed us to identify a four metal-thiolate cluster (alpha-domain) and a three metal-thiolate cluster (beta-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the alpha and beta domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the alpha-domain of mussel Cd(7)-MT10 is of the form M(4)S(12) instead of M(4)S(11), and has a novel topology. The beta-domain has a metal-thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the beta-domain displays a more disordered conformation than the alpha-domain. It is concluded that in mussel Cd(7)-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal-thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.
Department of Environmental and Life Sciences, University of Eastern Piedmont A. Avogadro, Via Bellini 25/G, Alessandria, 15100, Italy. giuseppe.digilio@mfn.unipmn.it
Giuseppe
G
Digilio
Chiara
C
Bracco
Laura
L
Vergani
Mauro
M
Botta
Domenico
D
Osella
Aldo
A
Viarengo
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D049878:Mytilus / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012015:Reference Standards
D007554:Isotopes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein