Toward ligand identification within a CCHHC zinc-binding domain from the NZF/MyT1 family.
A family of proteins that contain presumed zinc-binding domains with the consensus sequence Cys-X4-CysX4-His-X7-His-X5-Cys has recently been identified, but the metal binding and structural properties of these domains have not been investigated. This consensus is striking because of the presence of five conserved potential zinc-binding residues. A peptide corresponding to the third putative zinc-binding domain from the transcription factor NZF-1 (hereafter NZF-13) has been synthesized and characterized. The UV-visible absorption spectroscopic properties of the cobalt(H) complex of this peptide demonstrate that metal binding is tetrahedral, and the position of the visible absorption bands suggests coordination by three cysteinates and one histidine. To identify which of the two conserved histidine residues acts a metal-binding residue, two histidine to alanine variant peptides were also synthesized. Both variant peptides bound cobalt(II) in a tetrahedral fashion; replacement of the first of the two histidines has a somewhat larger effect on the detailed shape of the absorption spectral features than does replacement of the second histidine. These results suggest that the metal-coordinating residues (italicized) are Cys-X4-Cys-X4-His-X7-His-Xs-Cys. However, simultaneous substitution of both histidine residues with alanine generated a peptide with much more dramatically affected metal binding properties. These observations suggests that the relatively modest effects observed for the singly substituted peptides may be due to metal interactions involving the remaining histidine. Because of these phenomena, further studies will be required to establish more conclusively the roles of the two histidine residues in metal binding and the potential significance of the apparent alternative histidine coordination.
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
C A
CA
Blasie
J M
JM
Berg
D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification; D015534:Trans-Activators / Q000737:chemistry*; D016335:Zinc Fingers
D008024:Ligands; C099432:Myt1l protein, rat; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D015534:Trans-Activators