The Disassembly and Reassembly of Mutants of Escherichia Coli Heat-Labile Enterotoxin:  Replacement of Proline 93 Does Not Abolish the Reassembly-Competent and Reassembly-Incompetent States

Abstract

The carrier moiety of heat-labile enterotoxin of Escherichia coli (EtxB) is formed by the noncovalent association of identical monomeric subunits, which assemble, in vivo and in vitro, into exceptionally stable pentameric complexes. In vitro, acid disassembly followed by neutralization results in reassembly yields of between 20% and 60% depending on the identity of the salts present during the acid denaturation process. Loss of reassembly competence has been attributed to isomerization of the native cis-proline residue at position 93. To characterize this phenomenon further, two mutants of EtxB at proline 93 (P93G and P93A) were generated and purified. The proline variants reveal only minor differences in their biophysical and biochemical properties relative to wild-type protein, but major changes were observed in the kinetics of pentamer disassembly and reassembly. Additionally, a loss of assembly competence was observed following longer term acid treatment, which was even more marked than that of the wild-type protein. We present evidence that the loss of assembly competence of these mutants is best explained by a cis/trans peptidyl isomerization of the unfolded mutant subunits in acid conditions; this limited reassembly competence and the biophysical properties of the native P93 mutant pentamers imply the retention of the native cis conformation in the nonproline peptide bond between residues 92 and 93 in the mutated proteins.

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

“…The two toxins in vitro assemblies have also been reported [18], [19], [20], [21], [22]. Despite high sequence identity (84%), similar 3D structures and similar functions, the two toxins present different disassembly and reassembly features, even when performed under identical experimental conditions.…”

Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond

“…This residue is positioned in a reverse turn, immediately N-terminal of β6. The configuration of this proline residue has been shown to be important for proper toxin assembly [32].…”

“…are further known to catalyze disulfide bond formation in both toxin A (Cys187-Cys199) and B (Cys9-Cys86) subunits, which is required for their functional assembly [53,230,234,236,237]. In addition, a cis-proline at position 93 in the B subunits is necessary for efficient B pentamer formation [32]. A periplasmic peptidyl prolyl cis-trans isomerase is thought to catalyze this isomerization in vivo [32,223].…”

“…In addition, a cis-proline at position 93 in the B subunits is necessary for efficient B pentamer formation [32]. A periplasmic peptidyl prolyl cis-trans isomerase is thought to catalyze this isomerization in vivo [32,223]. These are the only proteins thought to be required for holotoxin assembly.…”

Targeting Cellular Receptors as a Strategy for the Development of New Generation Mucosal Vaccines

“…Cholera Toxin (CT) from Vibrio cholera and the heat-Labile Enterotoxin (LT) from Enterotoxigenic E. Coli (ETEC) are structurally related AB 5 enterotoxins, composed of an enzymatically active A subunit, which mediates their toxicity and a pentameric B subunit that binds with high affinity to ganglioside receptors expressed on the apical surface of M cells (Cheesman et al, 2004). Intranasal or oral immunization of mice with attenuated pathogens, or protective antigens, chemically or genetically conjugated to these toxins, or their respective B subunits, generated protection against subsequent lethal challenge with these pathogens (Jang et al, 2004;Bergquist et al, 1998;Rask et al, 2000).…”