title: Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit.
abstract: Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195-7420, USA.
authors.forename: F
authors.initials: F
authors.lastname: van den Akker
authors.forename: I K
authors.initials: IK
authors.lastname: Feil
authors.forename: C
authors.initials: C
authors.lastname: Roach
authors.forename: A A
authors.initials: AA
authors.lastname: Platas
authors.forename: E A
authors.initials: EA
authors.lastname: Merritt
authors.forename: W G
authors.initials: WG
authors.lastname: Hol
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry*; D001428:Bacterial Vaccines; D003460:Crystallization; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D004768:Enterotoxins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D015202:Protein Engineering
chemical_list: D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli