Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro.

Abstract

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly inl vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for preferentially excluding or favoring certain intermediates in the assembly of multisubunit proteins.

Role of an Intrasubunit Disulfide in the Association State of the Cytosolic Homo-oligomer Methionine Adenosyltransferase

“…To form this covalent bond, the residues must come to a distance such that the C␣ atoms of the cysteines are within 3.8 -4.5 Å of each other (9). These disulfides can be established within the same subunit, linking different areas of the polypeptide chain that may be quite distant in the sequence, or between subunits, binding each monomer on the right position to the others (10,11).…”

Formation of disulfide bonded dimer of mutated heat‐labile enterotoxin in vivo

“…Each monomer in the pentamer of B subunits contain an internal disulfide bond linking the cysteines at positions 9 and 86 [2]. When millimolar concentrations of the reducing agent, dithiothreitol (DTT), are added to the culture, newly synthesised B subunits are exported from the cytoplasm but pentamers do not form, although pentamers already made do not dissociate [3]. More surprising to us was our recent finding that the reduced monomers, which are hydrophilic after removal of the leader sequence, associate tightly with the membranes enclosing the periplasm, from which they can only be removed by washing with chaotropic agents [4].…”

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

“…Native cholera toxin is an AB 5 exotoxin composed of one catalytic A subunit (CtA) and 5 surface binding B subunits (Ctb) [21]. The A and B subunits spontaneously combine in a non-covalent fashion in the periplasm to form the holotoxin [22]. The 5 individual B subunits combine to form a ring-like structure (Ctb).…”