Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit.
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.
van den Akker
F
F
Department of Biochemistry, University of Washington, Seattle 98195-7420, USA.
Feil
I K
IK
Roach
C
C
Platas
A A
AA
Merritt
E A
EA
Hol
W G
WG
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
D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli