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 RAR␣ (retinoic acid receptor ␣) 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 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°C, and a ⌬G 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% ␤-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 I2 1 2 1 2 1 with a ‫؍‬ 38.8, b ‫؍‬ 77.7, and c ‫؍‬ 85.3 Å 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.The PLZF BTB/POZ domain, named for its presence in the Drosophila proteins Broad Complex, tramtrack, and bric a brac (BTB) (1) and its homology with several poxvirus proteins and zinc finger proteins (POZ) (2), is an evolutionarily conserved motif of about 120 residues (Fig. 1) found in an increasing number of proteins having a variety of functions (1-4). Proteins containing a BTB/POZ domain have been identified in poxvirus, Caenorhabditis elegans, Drosophila, and human and are generally found at the N terminus of either actin-binding or, more commonly, nuclear DNA-binding proteins (4). Proteins containing a BTB/POZ domain have been associated with diverse functions including nucleosome/chromosome disruption, pattern formation, metamorphosis, oogenesis, and eye and limb development (5-11).Biological relevance for the function of BTB/POZ domains has come from the study of the PLZF (for promyelocytic leukemia zinc finger) oncoprotein, associated with acute promyelocytic leukemia (APL).1 APL results from the malignant proliferation of cells blocked at the stage of promyelocytic differentiation and accounts for about 10% of all myeloid leukemias (...

In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions

“…Crystallographic analysis revealed that the POZ monomers interact via an extensive hydrophobic interface with interlocked ␣ helices and ␤ sheets, consistent with the observation that the domain is an obligate dimer (1,28). In addition, there is crystallographic evidence for higherorder associations between the BTB dimers through ␤-sheet interactions on the hydrophobic floor of the dimer (1,29).…”

KRIP6: A novel BTB/kelch protein regulating function of kainate receptors

“…Kelch superfamily proteins have diverse functions including regulating the actin cytoskeleton, gene expression, and protein ubiquitination. The BTB/POZ domain is a zinc finger and protein-protein interaction domain (Li et al, 1997) known to dimerize (e.g. (Cullen et al, 2004;Soltysik-Espanola et al, 1999).…”

The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT

“…First, the POZ domain can mediate homomeric as well as heteromeric POZ-POZ interactions (Bardwell and Treisman, 1994;Dhordain et al, 1995;Seyfert et al, 1996). The PLZF POZ domain, which has been studied in more detail, forms a very stable homodimer (Li et al, 1997b). Second, the POZ domains of BCL-6, PLZF and ZF5 have been shown to mediate transcriptional repression Albagli et al, 1996;Chang et al, 1996;Seyfert et al, 1996;Kaplan and Calame, 1997;Li et al, 1997a).…”