Metal and DNA binding properties of a two-domain fragment of neural zinc finger factor 1, a CCHC-type zinc binding protein.
Neural zinc finger factor 1 (NZF-1) is a member of a family of neural-specific transcription factors that contain multiple copies of a relatively uncharacterized zinc binding motif. We have studied the metal binding and DNA binding properties of a fragment of NZF-1 containing two adjacent zinc binding domains. Partial proteolysis with endoproteinase Lys-C identified metal-stabilized fragments containing either one or both of the zinc binding domains. Both domains were required for specific DNA binding to the beta-retinoic acid receptor element, producing a DNase I footprint covering predominantly one strand. The metal binding site was probed via cobalt(II) substitution. The visible absorption spectrum of the cobalt(II) complex is consistent with Cys-Cys-His-Cys coordination of the metal. The two domains appear to have similar affinities for metal and bind cobalt(II) and zinc(II) with dissociation constants of 4 (+/- 2) x 10(-)(7) M and 1.4 (+/- 0.8) x 10(-)(10) M, respectively. The domains fold upon the addition of zinc, as observed by (1)H NMR. However, an additional weak binding site causes line broadening in the presence of excess zinc, presumably due to aggregation.
Berkovits
H J
HJ
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Berg
J M
JM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D006868:Hydrolysis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000235:genetics; D020218:Response Elements; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
D004268:DNA-Binding Proteins; C099432:Myt1l protein, rat; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D018168:Receptors, Retinoic Acid; D015534:Trans-Activators; C082925:retinoic acid receptor beta; D003035:Cobalt; D003850:Deoxyribonuclease I; D015032:Zinc