Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b.
A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.
Gariépy
J
J
Department of Medicine, Stanford University School of Medicine, CA 94305.
Judd
A K
AK
Schoolnik
G K
GK
D000818:Animals; D000833:Animals, Suckling; D001427:Bacterial Toxins; D001681:Biological Assay; D003198:Computer Simulation; D003545:Cysteine; D003967:Diarrhea / Q000139:chemically induced; D004220:Disulfides; D004768:Enterotoxins; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase; D051379:Mice; D008958:Models, Molecular; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D003545:Cysteine