Solution structure of Cu₆ metallothionein from the fungus Neurospora crassa

The 3D-solution structure of Neurospora crassa Cu₆- metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional ¹H-NMR spectroscopy. It represents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a copper(I)-sulfur cluster. As seen with other MTs, the protein lacks definable secondary structural elements; however, the polypeptide fold is unique. The metal coordination and the cysteine spacing defines this unique fold. NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC-motif. This motif is found in a variety of mammalian MTs and metalloregulatory proteins. The in vitro formation of the Cu₆NcMT identical to the native Cu₆NcMT was dependent upon the prior formation of the Zn₃NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz ¹H-NMR spectral data permitted the 3D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as ¹¹³Cd and ¹⁰⁹Ag. These restraints have been necessary to establish specific metal to cysteine restraints in 3D structural studies on this family of proteins when using lower field, less sensitive ¹H-NMR spectral data. The accuracy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of the α-domain of mouse MT1 compared to the one recalculated without metal-cysteine connectivities.