Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein

The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface, A prototypic human BTB/POZ protein, PLZF (promyelocytic Leukemia zinc finger) is fused to RAR alpha (retinoic acid receptor alpha) in a subset of acute promyeloeytic leukemias (APLs), where it acts as a potent oncogene, The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a K-d below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding-transition with a T-m of 70.4 degrees C, and a Delta G of 12.8 +/- 0.4 kcal/mel. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet, The have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography, The crystals form in the space group I222 or I2(1)2(1)2(1) with a = 38.8, b = 77.7, and c = 85.3 Angstrom and contain 1 protein subunit per asymmetric unit, with approximately 40% solvent. Our data support the hypothesis that the ETB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function.