ASSEMBLY OF THE B-SUBUNIT PENTAMER OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN - KINETICS AND MOLECULAR-BASIS OF RATE-LIMITING STEPS IN-VITRO

The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homope ntameric complexes, which maintain their quaternary structure in a ran ge of conditions that would normally be expected to cause protein dena turation, 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. (19 95) J. Biol. Chem. 270, 29953-29958), Here, we investigate the influen ce of environmental parameters on the kinetics of reassembly of acid-g enerated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energ y of 76 +/- 2 J degrees mol(-1)degrees K-1, consistent with isomerizat ion of the cis-proline residue at position 93, and which prevented sub sequent EtxB reassembly. By using rapid neutralization of acid-generat ed monomers, a high proportion of the B-subunits adopted an assembly-c ompetent conformation, which resulted in up to 75% of the protein reas sembling into a stable pentameric complex, indistinguishable from nati ve EtxB pentamers. The rate-limiting step in reassembly, over a concen tration range of 50-200 mu g/ml, was shown 60 be due to an intramolecu lar event, which exhibited a pH dependence with a pK(a) of 7.0, Modifi cation of EtxB with amine-specific probes revealed that the protonatio n 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.