Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.

Abstract

Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains of Corynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT. This process is triggered by acidification from pH approximately 7 to pH approximately 5 within the endosome. Here we show that crm45 (cross-reacting material of 45 kDa), a 45 kDa deletion mutant of DT which contains the C and T domains but lacks the C-terminal receptor-binding (R) domain, undergoes a transition from a monomer to a large oligomer upon acidification from pH 7.0 to pH 5.0. Dynamic light scattering analysis of crm45 at pH 5.0 results in a polydispersity value of only 8-17%, suggesting that the oligomer is uniformly sized. Using analytical ultracentrifugation, measurements of the sedimentation rate and diffusion coefficient of crm45 at pH 5.0 result in a molecular mass determination of 890 +/- 40 kDa (20 +/- 1 subunits) for the oligomer. Equilibrium sedimentation data on crm45 at pH 5.0 are best fit by a single species with a mass of 1000 +/- 50 kDa (24 +/- 1 subunits). These results reveal the pH-dependent formation of a uniformly sized, 20-24 subunit oligomer of the C and T domains of DT, in solution. Because the oligomer of crm45 forms at the pH of the acidified endosome, it could be relevant to the translocation of the C domain of DT across the endosomal membrane and into the cytoplasm of host cells. The possible relevance of this oligomer of crm45 to the membrane translocation of the C domain of DT correlates with earlier kinetic studies of DT intoxication of Vero cells, which inferred the transfer of approximately 20 C domains of DT to the cytoplasm of host cells, in a single event.