Biochemical and Biophysical Properties of the Core-binding Factor α2 (AML1) DNA-binding Domain

Abstract

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 ␤ (CBF␤) subunit. In this study we overexpress the Runt domain from the mouse CBF␣2 (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 ␤-domain with essentially no ␣-helical content. The single tryptophan residue in the CBF␣2 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).

True

“…14,15 The AML1B DNA-binding domain has an ATP-binding site. 14,16 The physiologic role of CBF␣-ATP interactions remains to be determined. DNA-binding by the Runt domain is sensitive to oxidation, but the significance of this sensitivity is also unknown.…”

Mutagenesis of the Runt Domain Defines Two Energetic Hot Spots for Heterodimerization with the Core Binding Factor β Subunit

“…Urea Denaturation Monitored by Fluorescence Spectroscopy-We detected the urea denaturation of the Runt domain by tryptophan fluorescence as described previously (37). We performed all fluorescence experiments using a protein concentration of 20 -40 M in a total volume of 0.8 ml of 25 mM Tris-HCl (pH 7.5), 50 M EDTA, and 1 mM dithiothreitol on a SPEX Fluoromax spectrofluorometer (Spex Industries, Edison, NJ).…”

“…We labeled the wild type and mutated Runt domains (residues 41-214, numbered according to Bae et al (36)) with 15 N and purified them as described previously (23,37). We subcloned the CBF␤141 cDNA, which encodes the CBF␤ heterodimerization domain (amino acids 1-141), into the bacterial expression vector pET3c (Novagen) using NdeI and BamHI sites.…”

“…Therefore, for those mutants that showed altered NMR spectra, we also carried out urea denaturation measurements as an independent and quantitative measure of the effect of alanine substitution on protein stability (Table II). Denaturation with urea was monitored by means of the change in the fluorescence of the single tryptophan residue in the Runt domain, as described previously (37). Representative data for the wild type Runt domain and three of the mutant proteins are shown (Fig.…”

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

“…Following this, 4 μg/mL 3 ′ -A16-tagged RNA or 3 (Thornell et al 1991;Crute et al 1996;Wolf-Watz et al 1999) dissolved in buffer I was immobilized in flow cells 2-4 for 60 sec at a flow rate of 50 μL/min. RDE was used as a positive control and the 30N random RNA pool as a negative control.…”