Solution structure of Cu6 metallothionein from the fungus Neurospora crassa.
The 3D-solution structure of Neurospora crassa Cu(6)-metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional (1)H-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 Cu(6)NcMT identical to the native Cu(6)NcMT was dependent upon the prior formation of the Zn(3)NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz (1)H-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 (113)Cd and (109)Ag. 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 (1)H-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 alpha-domain of mouse MT1 compared to the one recalculated without metal-cysteine connectivities.
Cobine
Paul A
PA
Health Science Center, University of Utah, Salt Lake City, UT, USA.
McKay
Ryan T
RT
Zangger
Klaus
K
Dameron
Charles T
CT
Armitage
Ian M
IM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008668:Metallothionein / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D013455:Sulfur / Q000737:chemistry
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D008670:Metals; D010455:Peptides; C007121:copper thionein; D003553:Cystine; D013455:Sulfur; D003300:Copper; D008668:Metallothionein; D003545:Cysteine