SUPPRESSION OF TEMPERATURE-SENSITIVE ASSEMBLY MUTANTS OF HEAT-LABILE ENTEROTOXIN B-SUBUNITS

Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B subunit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42-degrees-C, the muta tions prevent the B subunits from achieving their final pentameric str ucture resulting in membrane association of the monomers. However, mut ant B subunits produced at 30-degrees-C assemble, in the periplasm, in to pentamers that remain stable when transferred to 42-degrees-C, indi cating that the mutant pentamers are stable under conditions where the ir formation is inhibited. The mutant pentamers are, similarly to wild -type pentamers, SDS-resistant and stable, in vitro, at temperatures u p to 65-degrees-C. This suggests that although the C-terminal amino ac ids are part of the subunit interface, they appear not to contribute s ignificantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly i ntermediate in the pentamerization process. Single second site mutatio ns suppress the assembly defect of mutant EtxB191.5, which carries sub stitutions at its C-terminus. The Thr --> Ile replacement at position 75 in the alpha2-helix probably restores the van der Waals contact bet ween residues 75 and 101, which had been greatly reduced by the Met -- > Leu substitution at position 101 in the beta6-strand of EtxB191.5. I nteraction between the alpha2-helix and beta6-strand which contains th e C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.