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 mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30 degrees C assemble, in the periplasm, into pentamers that remain stable when transferred to 42 degrees C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65 degrees C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr-->Ile replacement at position 75 in the alpha 2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met-->Leu substitution at position 101 in the beta 6-strand of EtxB191.5. Interaction between the alpha 2-helix and beta 6-strand which contains the C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.