IDENTIFICATION OF LYSINES WITHIN ALPHA-1-ANTICHYMOTRYPSIN IMPORTANT FOR DNA-BINDING - AN UNUSUAL COMBINATION OF DNA-BINDING ELEMENTS

被引:47
作者
NAIDOO, N
COOPERMAN, BS
WANG, ZM
LIU, XZ
RUBIN, H
机构
[1] UNIV PENN,DEPT CHEM,PHILADELPHIA,PA 19104
[2] UNIV PENN,DEPT MED,PHILADELPHIA,PA 19104
关键词
D O I
10.1074/jbc.270.24.14548
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 human serum serine protease inhibitor (serpin) alpha 1-antichymotrypsin (ACT) appears to be unique among serpins in its ability to bind to double-stranded DNA. Using site-directed mutagenesis and chemical modification, a tri-lysine sequence (residues 210-212) falling within a solvent exposed loop and the C-terminal peptide containing two lysines (residues 391 and 396) were shown to be important for DNA binding. Mutation of residues 210-212 from lysines to either glutamates or threonines abolished DNA binding. The Lys(210)-Thr(211)-Lys(212) and Thr(210)-Th4(211)-Lys(212) variants displayed reduced affinity for DNA, especially at higher ionic strength. Limited acetylation of rACT with acetic anhydride led to loss of DNA binding and, conversely, DNA protected rACT from acetylation. A combination of CNBr digestion, peptide separation, and peptide sequencing identified Lys(396), two residues from the C terminus, as the most reactive lysine in rACT. Acetylation of Lys(396) is strongly decreased in the presence of DNA. The double mutant K391T/K396T-rACT had very little affinity for DNA. The epsilon-amines of lysines 210-212 are 8-15 Angstrom across a cleft from the epsilon-amines in Lys(391) adn Lys(396), and together these two elements may form an unusual DNA binding domain. Attempts to isolate a DNA sequence to which ACT binds specifically have been unsuccessful to date, raising the possibility that nonspecific binding of ACT to DNA suffices to account for the ACT found in certain cell nuclei. ACT variants not binding to double stranded DNA retain ACT protease inhibitory activity, a potentially important result for the use of ACT variants as therapeutic agents.
引用
收藏
页码:14548 / 14555
页数:8
相关论文
共 56 条
  • [1] IMMUNOCHEMICAL IDENTIFICATION OF THE SERINE PROTEASE INHIBITOR ALPHA-1-ANTICHYMOTRYPSIN IN THE BRAIN AMYLOID DEPOSITS OF ALZHEIMERS-DISEASE
    ABRAHAM, CR
    SELKOE, DJ
    POTTER, H
    [J]. CELL, 1988, 52 (04) : 487 - 501
  • [2] THE ROLE OF THE ACUTE-PHASE PROTEIN ALPHA-1-ANTICHYMOTRYPSIN IN BRAIN-DYSFUNCTION AND INJURY
    ABRAHAM, CR
    [J]. RESEARCH IN IMMUNOLOGY, 1992, 143 (06): : 631 - 636
  • [3] ADDISON C, 1990, ONCOGENE, V5, P423
  • [4] MOLECULAR EVOLUTION OF SERPINS - HOMOLOGOUS STRUCTURE OF THE HUMAN ALPHA-1-ANTICHYMOTRYPSIN AND ALPHA-1-ANTITRYPSIN GENES
    BAO, JJ
    SIFERS, RN
    KIDD, VJ
    LEDLEY, FD
    WOO, SLC
    [J]. BIOCHEMISTRY, 1987, 26 (24) : 7755 - 7759
  • [5] CRYSTAL-STRUCTURE OF CLEAVED HUMAN ALPHA-1-ANTICHYMOTRYPSIN AT 2.7-A RESOLUTION AND ITS COMPARISON WITH OTHER SERPINS
    BAUMANN, U
    HUBER, R
    BODE, W
    GROSSE, D
    LESJAK, M
    LAURELL, CB
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1991, 218 (03) : 595 - 606
  • [6] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [7] PROTEIN ESTIMATION BY THE PRODUCT OF INTEGRATED PEAK AREA AND FLOW-RATE
    BUCK, MA
    OLAH, TA
    WEITZMANN, CJ
    COOPERMAN, BS
    [J]. ANALYTICAL BIOCHEMISTRY, 1989, 182 (02) : 295 - 299
  • [8] SEQUENCE HOMOLOGY BETWEEN HUMAN ALPHA-1-ANTICHYMOTRYPSIN, ALPHA-1-ANTITRYPSIN, AND ANTI-THROMBIN-III
    CHANDRA, T
    STACKHOUSE, R
    KIDD, VJ
    ROBSON, KJH
    WOO, SLC
    [J]. BIOCHEMISTRY, 1983, 22 (22) : 5055 - 5060
  • [9] THE DNA-BINDING ARM OF LAMBDA-REPRESSOR - CRITICAL CONTACTS FROM A FLEXIBLE REGION
    CLARKE, ND
    BEAMER, LJ
    GOLDBERG, HR
    BERKOWER, C
    PABO, CO
    [J]. SCIENCE, 1991, 254 (5029) : 267 - 270
  • [10] COOPERMAN BS, 1993, J BIOL CHEM, V268, P23626