Solution properties of the free and DNA-bound Runt domain of AML1

The Punt domain is responsible for specific DNA and protein-protein interac tions in a family of transcription factors which includes human AML1. Struc tural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here w e describe the optimization and characterization of a 140-residue fragment, containing the Punt domain of AML1, which is suitable for structural studi es. The fragment of AML1 including amino acids 46-185 [AML1(DM)(46-185)] co ntains a double cysteine-->serine mutation which does not affect Runt domai n structure or DNA-binding affinity. Purified AML1 (DM)(46-185) is soluble and optimally stable in a buffer containing 200 mM MgSO4 and 20 mM sodium p hosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spect roscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-speci fic DNA binding. The NMR spectrum of AML1 (DM)(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily iden tified, suggesting that a structure determination of this Runt domain fragm ent is feasible. A titration of N-15-labeled AML1 (DM)(46-185) with a 14-bp cognate DNA duplex results in changes in the N-15 NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific comp lex and structural changes in the Punt domain upon DNA binding.