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

被引:60
作者
Li, XM
LopezGuisa, JM
Ninan, N
Weiner, EJ
Rauscher, FJ
Marmorstein, R
机构
[1] UNIV PENN,WISTAR INST,PHILADELPHIA,PA 19104
[2] UNIV PENN,DEPT CHEM,PHILADELPHIA,PA 19104
[3] UNIV PENN,DEPT BIOCHEM & BIOPHYS,PHILADELPHIA,PA 19104
关键词
D O I
10.1074/jbc.272.43.27324
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:27324 / 27329
页数:6
相关论文
共 37 条
  • [1] ALBAGLI O, 1995, CELL GROWTH DIFFER, V6, P1193
  • [2] THE POZ DOMAIN - A CONSERVED PROTEIN-PROTEIN INTERACTION MOTIF
    BARDWELL, VJ
    TREISMAN, R
    [J]. GENES & DEVELOPMENT, 1994, 8 (14) : 1664 - 1677
  • [3] ANTISENSE OLIGONUCLEOTIDES FROM THE STAGE-SPECIFIC MYELOID ZINC FINGER GENE MZF-1 INHIBIT GRANULOPOIESIS INVITRO
    BAVISOTTO, L
    KAUSHANSKY, K
    LIN, N
    HROMAS, R
    [J]. JOURNAL OF EXPERIMENTAL MEDICINE, 1991, 174 (05) : 1097 - 1101
  • [4] BROOKS I, 1994, METHOD ENZYMOL, V240, P459
  • [5] BUJARD H, 1987, METHOD ENZYMOL, V155, P416
  • [6] CHEN W, 1995, MOL CELL BIOL, V15, P3424
  • [7] PLZF-RAR-ALPHA FUSION PROTEINS GENERATED FROM THE VARIANT T(11-17)(Q23-Q21) TRANSLOCATION IN ACUTE PROMYELOCYTIC LEUKEMIA INHIBIT LIGAND-DEPENDENT TRANSACTIVATION OF WILD-TYPE RETINOIC ACID RECEPTORS
    CHEN, Z
    GUIDEZ, F
    ROUSSELOT, P
    AGADIR, A
    CHEN, SJ
    WANG, ZY
    DEGOS, L
    ZELENT, A
    WAXMAN, S
    CHOMIENNE, C
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (03) : 1178 - 1182
  • [8] FUSION BETWEEN A NOVEL KRUPPEL-LIKE ZINC FINGER GENE AND THE RETINOIC ACID RECEPTOR-ALPHA LOCUS DUE TO A VARIANT T(11,17) TRANSLOCATION ASSOCIATED WITH ACUTE PROMYELOCYTIC LEUKEMIA
    CHEN, Z
    BRAND, NJ
    CHEN, A
    CHEN, SJ
    TONG, JH
    WANG, ZY
    WAXMAN, S
    ZELENT, A
    [J]. EMBO JOURNAL, 1993, 12 (03) : 1161 - 1167
  • [9] Cudney R, 1994, ACTA CRYSTALLOGR D, V50, P414
  • [10] DIBELLO PR, 1991, GENETICS, V129, P385