title: Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.
abstract: The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homopentameric complexes, which maintain their quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. Recently, we showed that the simultaneous protonation of two of the COOH-terminal carboxylates in pentameric EtxB was required to cause its disassembly at pH values below 2.0 (Ruddock, L., Ruston, S. P., Kelly, S. M., Price, N. C., Freedman, R. B., and Hirst, T. R.(1995) J. Biol. Chem. 270, 29953-29958). Here, we investigate the influence of environmental parameters on the kinetics of reassembly of acid-generated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energy of 76 +/- 2 J.mol-1.K-1, consistent with isomerization of the cis-proline residue at position 93, and which prevented subsequent EtxB reassembly. By using rapid neutralization of acid-generated monomers, a high proportion of the B-subunits adopted an assembly-competent conformation, which resulted in up to 75% of the protein reassembling into a stable pentameric complex, indistinguishable from native EtxB pentamers. The rate-limiting step in reassembly, over a concentration range of 50-200 microg/ml, was shown to be due to an intramolecular event, which exhibited a pH dependence with a pKa of 7.0. Modification of EtxB with amine-specific probes revealed that the protonation state of the NH2-terminal alanine residue was responsible for the pH dependence of reassembly. The implications of these findings for the biogenesis of Escherichia coli enterotoxin and related enterotoxins in vivo, are considered.
authors.affiliation: Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom.
authors.forename: L W
authors.initials: LW
authors.lastname: Ruddock
authors.forename: J J
authors.initials: JJ
authors.lastname: Coen
authors.forename: C
authors.initials: C
authors.lastname: Cheesman
authors.forename: R B
authors.initials: RB
authors.lastname: Freedman
authors.forename: T R
authors.initials: TR
authors.lastname: Hirst
mesh_terms: D000284:Administration, Oral; D000941:Antigens / Q000008:administration & dosage; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D004337:Drug Carriers; D004768:Enterotoxins / Q000737:chemistry* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D013696:Temperature
chemical_list: D000941:Antigens; D001427:Bacterial Toxins; D004337:Drug Carriers; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli