title: Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein.
abstract: 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 RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic 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 Kd 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 Tm of 70.4 degrees C, and a DeltaG of 12.8 +/- 0.4 kcal/mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. We 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 I212121 with a = 38.8, b = 77.7, and c = 85.3 A and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/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.
authors.affiliation: The Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: J M
authors.initials: JM
authors.lastname: Lopez-Guisa
authors.forename: N
authors.initials: N
authors.lastname: Ninan
authors.forename: E J
authors.initials: EJ
authors.lastname: Weiner
authors.forename: F J
authors.initials: FJ
authors.lastname: Rauscher
authors.forename: R
authors.initials: R
authors.lastname: Marmorstein
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D017124:Conserved Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D019281:Dimerization; D029721:Drosophila Proteins; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014461:Ultracentrifugation; D016335:Zinc Fingers
chemical_list: C093752:Bric-a-brac protein 1, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D014157:Transcription Factors; C090212:ZBTB6 protein, human; C092037:ZBTB16 protein, human