The formation and structure of Escherichia coli K-12 haemolysin E pores

Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Correspondence Jeffrey Green jeff.green{at}sheffield.ac.uk Some enteric bacteria synthesize a pore-forming toxin, HlyE, which is cytolytic and cytotoxic to host cells. Measurement of HlyE binding to erythrocyte ghosts and the kinetics of HlyE-mediated erythrocyte lysis suggests that interaction with target membranes is not the rate-limiting step in the formation of HlyE pores, but that there is a temperature-dependent lag phase before a functional pore is formed. Circular dichroism and fluorescence energy transfer analyses show that HlyE protomers retain an -helical structure when oligomerized to form a pore consisting of parallel HlyE protomers. Comparison of the proteolytic sensitivities of the water-soluble and oligomeric forms of HlyE identifies inner and outer surfaces of the pore. This new information has been used to constrain a model of the HlyE pore, which allows a more detailed interpretation of previous low-resolution 3D reconstructions and suggests a novel mechanism for insertion of HlyE into target membranes. Abbreviations: β -OG, n -octyl β - D -glucopyranoside Two supplementary figures of models of octameric and 13-meric HlyE pores showing proteolytic cleavage sites and of the locations of proteolytic sites mapped onto an HlyE protomer are available with the online version of this paper.