Solution structure of Cu6 metallothionein from the fungus Neurospora crassa

Paul A. Cobine, Ryan T. McKay, Klaus Zangger, Charles T. Dameron, Ian M. Armitage

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4213-4221
Number of pages9
JournalEuropean Journal of Biochemistry
Volume271
Issue number21
DOIs
StatePublished - Nov 2004

Keywords

  • Copper
  • Metallothionein
  • NMR
  • Neurospora crassa
  • Solution structure

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