Intermolecular interactions between the A and B subunits of heat-labile enterotoxin from Escherichia coli promote holotoxin assembly and stability in vivo.
Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex.
Streatfield
S J
SJ
Biological Laboratory, University of Kent, Canterbury, Great Britain.
Sandkvist
M
M
Sixma
T K
TK
Bagdasarian
M
M
Hol
W G
WG
Hirst
T R
TR
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli