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 AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is 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 We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue.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 contains 103 residues for each of the five B subunits, 175 residues for the A1 subunit, and 41 residues for the A2 subunit. In this Arg7Lys structure the active site cleft within the A subunit is wider by approximately 1 A than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in the 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 differences observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required for full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
van den Akker
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Department of Biological Structure and Biochemistry, University of Washington, Seattle, USA.
Merritt
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Pizza
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Domenighini
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Rappuoli
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Hol
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WG
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D009243:NAD; C027538:heat-labile enterotoxin, E coli