Functional characterization of iron-substituted neural zinc finger factor 1: metal and DNA binding.
Neural zinc finger factor 1 (NZF-1) is a nonclassical zinc finger protein involved in neuronal development. NZF-1 contains multiple copies of a unique CCHHC zinc-binding domain that recognize a promoter element in the beta-retinoic acid receptor gene termed beta-retinoic acid receptor element (beta-RARE). Previous studies have established that a two-domain fragment of NZF-1 bound with zinc is sufficient for specific DNA binding. Proper functioning of the nervous system relies heavily on iron and misregulation of this highly redox active metal has serious consequences. Several classes of zinc finger proteins have been shown to bind other metal ions, including iron. To determine if ferrous iron can coordinate to the metal-binding sites of NZF-1 and assess the functional consequences of such coordination, a fragment of NZF-1 that contains two zinc-binding domains, NZF-1 double finger (NZF-1-DF), was prepared. UV-vis spectroscopy experiments demonstrated that Fe(II) is capable of binding to NZF-1-DF. Upon reconstitution with either Fe(II) or Zn(II), NZF-1-DF binds selectively and tightly (nanomolar affinity) to its target beta-RARE DNA sequence, whereas apo-NZF-1-DF does not bind to DNA and instead aggregates.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.
Angelique N
AN
Besold
Seung Jae
SJ
Lee
Sarah L J
SL
Michel
Niall Lue
NL
Sue
Holly J
HJ
Cymet
D000388:Air; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003300:Copper / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D007501:Iron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001059:Apoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D003300:Copper; D004247:DNA; D007501:Iron