title: Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity.
abstract: Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.
authors.affiliation: Howard Hughes Medical Institute, University of Washington, Seattle 98195-7742, USA.
authors.forename: I K
authors.initials: IK
authors.lastname: Feil
authors.forename: R
authors.initials: R
authors.lastname: Reddy
authors.forename: L
authors.initials: L
authors.lastname: de Haan
authors.forename: E A
authors.initials: EA
authors.lastname: Merritt
authors.forename: F
authors.initials: F
authors.lastname: van den Akker
authors.forename: D R
authors.initials: DR
authors.lastname: Storm
authors.forename: W G
authors.initials: WG
authors.lastname: Hol
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005260:Female; D016297:Mutagenesis, Site-Directed; D010053:Ovary / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli; D000242:Cyclic AMP