ATP-dependent degradation of CcdA by Lon protease - Effects of secondary structure and heterologous subunit interactions

被引:174
作者
VanMelderen, L
Thi, MHD
Lecchi, P
Gottesman, S
Couturier, M
Maurizi, MR
机构
[1] NCI,CELL BIOL LAB,BETHESDA,MD 20892
[2] NCI,MOL BIOL LAB,BETHESDA,MD 20892
[3] FREE UNIV BRUSSELS,DEPT BIOL MOL,GENET LAB,B-1640 RHODE ST GENESE,BELGIUM
[4] FREE UNIV BRUSSELS,INST MOL BIOL,B-1640 RHODE ST GENESE,BELGIUM
[5] NIDDK,ANALYT CHEM LAB,NIH,BETHESDA,MD 20892
关键词
D O I
10.1074/jbc.271.44.27730
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
CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (K-m greater than or equal to 200 mu M), and the peptide bond turnover number was similar to 10 min(-1). CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity, CcdA lost alpha-helical structure at elevated temperatures (T-m similar to 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature, ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.
引用
收藏
页码:27730 / 27738
页数:9
相关论文
共 40 条
  • [1] CELL KILLING BY THE F-PLASMID CCDB PROTEIN INVOLVES POISONING OF DNA-TOPOISOMERASE-II COMPLEXES
    BERNARD, P
    COUTURIER, M
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1992, 226 (03) : 735 - 745
  • [2] THE F-PLASMID CCDB PROTEIN INDUCES EFFICIENT ATP-DEPENDENT DNA CLEAVAGE BY GYRASE
    BERNARD, P
    KEZDY, KE
    VANMELDEREN, L
    STEYAERT, J
    WYNS, L
    PATO, ML
    HIGGINS, PN
    COUTURIER, M
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1993, 234 (03) : 534 - 541
  • [3] THE 41 CARBOXY-TERMINAL RESIDUES OF THE MINIF PLASMID CCDA PROTEIN ARE SUFFICIENT TO ANTAGONIZE THE KILLER ACTIVITY OF THE CCDB PROTEIN
    BERNARD, P
    COUTURIER, M
    [J]. MOLECULAR & GENERAL GENETICS, 1991, 226 (1-2): : 297 - 304
  • [4] Chou P Y, 1978, Adv Enzymol Relat Areas Mol Biol, V47, P45
  • [5] AUTO-REGULATION OF THE CCD OPERON IN THE F-PLASMID
    DEFEYTER, R
    WALLACE, C
    LANE, D
    [J]. MOLECULAR & GENERAL GENETICS, 1989, 218 (03): : 481 - 486
  • [6] MATRIX SELECTION FOR LIQUID SECONDARY-ION AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY
    DEPAUW, E
    [J]. METHODS IN ENZYMOLOGY, 1990, 193 : 201 - 214
  • [7] GERDES K, 1990, New Biologist, V2, P946
  • [8] GOLDBERG AL, 1985, J BIOL CHEM, V260, P2029
  • [9] THE MECHANISM AND FUNCTIONS OF ATP-DEPENDENT PROTEASES IN BACTERIAL AND ANIMAL-CELLS
    GOLDBERG, AL
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1992, 203 (1-2): : 9 - 23
  • [10] THE FACTOR-F OF ESCHERICHIA-COLI CARRIES A LOCUS OF STABLE PLASMID INHERITANCE STM, SIMILAR TO THE PARB LOCUS OF PLASMID-RI
    GOLUB, EI
    PANZER, HA
    [J]. MOLECULAR & GENERAL GENETICS, 1988, 214 (02): : 353 - 357