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

doi:10.1074/jbc.271.42.26251

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

From the Departments of ^‡ Biochemistry,
^¶ Pharmacology and Toxicology, and
^∥ Chemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

^″ Supported in part by a grant from the Leukemia Research Foundation and by a grant from the Human Frontier Science Program Organization (J. P. Gergen, P.I.). To whom correspondence should be addressed. Tel.: 603-646-1567; Fax: 603-646-3943.
^‴ A Leukemia Society of America Scholar. Supported in part by United States Public Health Service Grant CA58343 from the National Cancer Institute and by a grant from the Human Frontier Science Program Organization (J. P. Gergen, P.I.). To whom correspondence should be addressed. Tel.: 603-650-1159; Fax: 603-650-1128.

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 β (CBFβ) subunit. In this study we overexpress the Runt domain from the mouse CBFα2 (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 β-domain with essentially no α-helical content. The single tryptophan residue in the CBFα2 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).

Footnotes

^§ Supported by National Institutes of Health Training Grants AI07363 and CA09658.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CBF

core-binding factor

PCR

polymerase chain reaction

DTT

dithiothreitol

HA

high affinity

WT

wild type

PAGE

polyacrylamide gel electrophoresis

RU

response unit

NATA

N-acetyltryptophanamide.
↵² B. E. Crute, X. Huang, N. A. Speck, and J. H. Bushweller, unpublished results.
↵³ Y. Akamatsu and K. Shigesada, personal communication.
↵⁴ X. Huang, unpublished results.
↵⁵ B. E. Crute, A. F. Lewis, and N. A. Speck, unpublished results.
↵⁶ A. F. Lewis and N. A. Speck, unpublished results.
- Received May 17, 1996.
- Revision received August 1, 1996.