Biochemical and biophysical properties of the core-binding factor alpha2 (AML1) DNA-binding domain.
The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes. Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals. In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit. In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction. We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology. Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content. The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment. Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).
Crute
B E
BE
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Lewis
A F
AF
Wu
Z
Z
Bushweller
J H
JH
Speck
N A
NA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D050676:Core Binding Factor Alpha 2 Subunit; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D004926:Escherichia coli; D051379:Mice; D009687:Nuclear Proteins; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan
D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C493726:Runx1 protein, mouse; D014157:Transcription Factors; C080833:run protein, Drosophila; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004247:DNA