RESUMO
Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Receptores do Ácido Retinoico/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Ressonância de Plasmônio de Superfície , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains.