Nature Structural Biology9, 729 - 733 (2002)
Published online: 9 September 2002; | doi:10.1038/nsb839
Conversion of a transmembrane to a water-soluble protein complex by a single point mutation
Yulia Tsitrin1, Craig J. Morton2, Catherine El Bez3, Patrick Paumard1, Marie-Claire Velluz1, Marc Adrian3, Jacques Dubochet3, Michael W. Parker2, 5, Salvatore Lanzavecchia4
& F.G. van der Goot1, 5
1
Department of Genetics and Microbiology, University of Geneva, 1 rue Michel Servet, 1211 Geneva, Switzerland.
2
The Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
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.
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