THE ARG7LYS MUTANT OF HEAT-LABILE ENTEROTOXIN EXHIBITS GREAT FLEXIBILITY OF ACTIVE-SITE LOOP-47-56 OF THE A-SUBUNIT

The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB(5) bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including G(5 alpha). The A subunit i s responsible for this covalent modification, while the B pentamer is involved in receptor recognition. We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue. The final model contain s 103 residues for each of the five B subunits, 175 residues for the A (1) subunit, and 41 residues for the A(2) subunit. In this Arg7Lys str ucture the active site cleft within the A subunit is wider by approxim ately 1 Angstrom than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in th e Arg7Lys structure, even though the new lysine residue at position 7 assumes a position which virtually coincides with that of Arg7 in the wild-type structure. The displacement of residues 47-56 as seen in the mutant structure is proposed to be necessary for allowing NAD access to the active site of the wild-type LT. On the basis of the difference s observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required fo r full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subun it. These proposed conformational changes start at the site of reducti on and cleavage and propagate a distance of over 20 Angstrom to affect the active site itself.