Organization of diphtheria toxin in membranes - A hydrophobic photolabeling study

被引:28
作者
D'Silva, PR
Lala, AK [1 ]
机构
[1] Indian Inst Technol, Dept Chem, Biomembrane Lab, Bombay 400076, Maharashtra, India
[2] Indian Inst Technol, Ctr Biotechnol, Bombay 400076, Maharashtra, India
关键词
D O I
10.1074/jbc.275.16.11771
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
Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH (similar to 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134-141, originally corresponding to a beta-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.
引用
收藏
页码:11771 / 11777
页数:7
相关论文
共 58 条
[1]  
AMES BN, 1960, J BIOL CHEM, V235, P769
[2]   Groups with polar characteristics can locate at both shallow and deep locations in membranes: The behavior of dansyl and related probes [J].
Asuncion-Punzalan, E ;
Kachel, K ;
London, E .
BIOCHEMISTRY, 1998, 37 (13) :4603-4611
[3]   REFINED STRUCTURE OF MONOMERIC DIPHTHERIA-TOXIN AT 2.3-ANGSTROM RESOLUTION [J].
BENNETT, MJ ;
EISENBERG, D .
PROTEIN SCIENCE, 1994, 3 (09) :1464-1475
[4]   THE MOLTEN GLOBULE STATE IS INVOLVED IN THE TRANSLOCATION OF PROTEINS ACROSS MEMBRANES [J].
BYCHKOVA, VE ;
PAIN, RH ;
PTITSYN, OB .
FEBS LETTERS, 1988, 238 (02) :231-234
[5]   THE CRYSTAL-STRUCTURE OF DIPHTHERIA-TOXIN [J].
CHOE, S ;
BENNETT, MJ ;
FUJII, G ;
CURMI, PMG ;
KANTARDJIEFF, KA ;
COLLIER, RJ ;
EISENBERG, D .
NATURE, 1992, 357 (6375) :216-222
[6]   DIPHTHERIA-TOXIN - MODE OF ACTION AND STRUCTURE [J].
COLLIER, RJ .
BACTERIOLOGICAL REVIEWS, 1975, 39 (01) :54-85
[7]   Unfolding of diphtheria toxin - Identification of hydrophobic sites exposed on lowering of pH by photolabeling [J].
D'Silva, PR ;
Lala, AK .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (26) :16216-16222
[8]   Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates [J].
D'Silva, PR ;
Lala, AK .
PROTEIN SCIENCE, 1999, 8 (05) :1099-1103
[9]  
EIBL H, 1983, METHOD ENZYMOL, V98, P623
[10]  
GONZALEZ JE, 1988, J BIOL CHEM, V263, P15257