Conversion of a transmembrane to a water-soluble protein complex by a single point mutation

Abstract

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.

Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism

“…Importantly, K246C-E258C heptamers are partly hydrophobic, as opposed to Y221G heptamers, indicating that they are blocked beyond the prepore stage 8 .…”

“…1), is blocked at the prepore stage and thus forms soluble heptamers 8 . The second, K246C-E258C (inset in Fig.…”

“…forming the transmembrane portion is again incompatible with the initial model, where domain 4 is transmembrane 4,8 .…”

“…We previously analyzed Y221G soluble heptamers by single particle cryo-EM on negatively stained samples 8 . Here we performed cryo-EM on Y221G heptamers without stain to obtain a more accurate model.…”

From Tilings to Fibers – Bio-mathematical Aspects of Fold Plasticity

“…Likewise the pore forming toxin aerolysin can form a dimer of heptamer after a single amino acid mutation (see [33]). It remains to be established whether the dimeric versions of the pentamer and heptamer are more prompt to form fibers than their single oligomeric counterpart.…”