Solution Properties Of The Free And Dna-Bound Runt Domain Of Aml1

The Punt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Punt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1(DM)(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain 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 phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy 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-specific DNA binding. The NMR spectrum of AML1 (DM)(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment 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 complex and structural changes in the Punt domain upon DNA binding.