Modulation of DNA binding of a tramtrack zinc finger peptide by the metallothionein-thionein conjugate pair.
The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of alpha-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of alpha-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.
Roesijadi
G
G
University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland 20688, USA. groes@cbl.umces.edu
Bogumil
R
R
Vasák
M
M
Kägi
J H
JH
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029721:Drosophila Proteins; D007700:Kinetics; D008668:Metallothionein / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D012097:Repressor Proteins; C075878:ttk protein, Drosophila; D004247:DNA; D008668:Metallothionein