4212–4222 Nucleic Acids Research, 2005, Vol. 33, No. 13 doi:10.1093/nar/gki724 Cooperative binding of DNA and CBFb to the Runt domain of the CBFa studied via MD simulations Bahru Habtemariam, Victor M. Anisimov and Alexander D. MacKerell, Jr* Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA Received May 27, 2005; Revised and Accepted July 5, 2005 ABSTRACT The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor b (CBFb) also binds to the RD to enhance RD–DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFb and in a ternary complex with DNA and CBFb. Consistent with the experimental findings, in the presence of CBFb the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermol- ecular interactions and desolvation contributions. Also contributing to the enhanced binding are favor- able intramolecular interactions between the ‘wing’ residues (RD residues 139–145) and the ‘wing1’ residues (RD residues 104–116). The simulation stud- ies also indicate that the RD–CBFb binding is more favorable in the presence of DNA due to a more favorable RD–CBFb interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to inter- pret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD–CBFb interaction is predicted. INTRODUCTION The Runt domain (RD) is the DNA-binding domain of p53 type transcription factors known as core binding factor a (CBFa) subunits. The CBFas are expressed by the three related genes Runx1, Runx2 and Runx3 (1). The three Runx genes are important for developmental processes; Runx1 is important for hematopoiesis, Runx2 is essential for skeletal development (osteogenesis) and Runx3 is required for the development of gastric epithelium (2–4). Runx1 and Runx2 also have been shown to be important for vascular development or angio- genesis and their activity is augmented by angiogenic growth factors (5,6). Mutation of the Runx1 gene is associated with human leukemias, whereas mutation of the Runx3 gene is involved with the development and progression of gastric can- cers and testicular yolk sac tumor (1,7,8). Mutation of the Runx2 gene is associated with the inherited human skeletal disorder known as cleidocranial dysplasia (9). A related pro- tein, known as CBFb, binds to the RD of the CBFas and enhances the DNA-binding afﬁnity of the RD. The RD is the site of both DNA binding and heterodimerization with CBFb. Because of the involvement of CBFas in many developmental processes and cancerous diseases, understanding RD–DNA and RD–CBFb interactions may shed light on the control of these processes. In addition, the involvement of Runx genes with angiogenesis also makes the RD a potential target in the development and treatment of many types of cancers. The 3D structures of the RD in the free state, bound to DNA, CBFb and both DNA and CBFb have been determined using nuclear magnetic resonance (NMR) spectroscopy and/or X-ray crystallography (10–14). Biochemical and biophysical studies have been performed characterizing the binding of RD to DNA in the presence and absence of CBFb (15,16). In the presence of CBFb, the dissociation constant (KD) for RD–DNA decreases 6- to 10-fold (16). Several studies have identiﬁed RD residues important for both DNA and CBFb binding via mutational analysis (17–19). The mutational analysis usually involved mutations to alanine and analysis of its effect on RD–DNA and RD–CBFb-binding activities, as determined using yeast hybrid assay analysis. In addition, a recent NMR-based study has identiﬁed a variety of residues involved in the cooperativity associated with CBFb binding to RD (20). While these important studies have given insights into the role of individual residues to RD–DNA and RD–CBFb interactions, the interpretation of the mutational analysis may be complicated by the inﬂuence of the mutation on protein stability, protein folding and other localized changes, espe- cially at protein–protein interfaces, while structural and dynamic information from NMR and X-ray studies cannot yield energetic information at an atomic level of detail. *To whom correspondence should be addressed at 20 Penn Street, Baltimore, MD 21201, USA. 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