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PLZF癌蛋白中BTB/POZ结构域的过表达、纯化、特性鉴定及结晶

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

作者信息

Li X, Lopez-Guisa J M, Ninan N, Weiner E J, Rauscher F J, Marmorstein R

机构信息

The Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

出版信息

J Biol Chem. 1997 Oct 24;272(43):27324-9. doi: 10.1074/jbc.272.43.27324.

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.

摘要

BTB/POZ结构域定义了一个约120个残基的保守区域,迄今为止已在40多种蛋白质中发现。它主要位于锌指DNA结合蛋白的N端,在那里它可能作为一个抑制结构域发挥作用,而在肌动蛋白结合蛋白和痘病毒编码蛋白中出现的频率较低,在那里它可能作为一个蛋白质-蛋白质相互作用界面发挥作用。一种典型的人类BTB/POZ蛋白,早幼粒细胞白血病锌指蛋白(PLZF),在一部分急性早幼粒细胞白血病(APL)中与视黄酸受体α(RARα)融合,在那里它作为一种强效癌基因发挥作用。BTB/POZ结构域在蛋白质-蛋白质相互作用和/或转录调控中的确切作用尚不清楚。我们已经对PLZF的BTB/POZ结构域(PLZF-BTB/POZ)进行了过表达、纯化、表征和结晶。凝胶过滤、动态光散射和平衡沉降实验表明,PLZF-BTB/POZ形成了一个Kd低于200 nM的同二聚体。差示扫描量热法和平衡变性实验表明,PLZF-BTB/POZ二聚体经历了一个两态展开转变,Tm为70.4℃,ΔG为12.8±0.4 kcal/mol。圆二色性表明,PLZF-BTB/POZ二聚体具有显著的二级结构,包括约45%的螺旋和20%的β-折叠。我们已经制备了适合于使用X射线晶体学进行高分辨率结构测定的PLZF-BTB/POZ晶体。晶体在空间群I222或I212121中形成,a = 38.8,b = 77.7,c = 85.3 Å,每个不对称单元包含1个蛋白质亚基,溶剂含量约为40%。我们的数据支持这样一种假设,即BTB/POZ结构域在体内介导一种功能相关的二聚化功能。PLZF-BTB/POZ结构域的晶体结构将为理解BTB/POZ结构域功能的结构基础提供一个范例。

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