The formation and structure of Escherichia coli K-12 haemolysin E pores.
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 alpha-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.
Hunt
Stuart
S
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Moir
Arthur J G
AJG
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Tzokov
Svetomir
S
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Bullough
Per A
PA
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Artymiuk
Peter J
PJ
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Green
Jeffrey
J
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004912:Erythrocytes / Q000382:microbiology; D048168:Escherichia coli K12 / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005470:Fluorometry; D006460:Hemolysin Proteins / Q000737:chemistry*; D006461:Hemolysis; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008958:Models, Molecular; D015394:Molecular Structure; D018272:Porins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013696:Temperature
D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D018272:Porins; C119944:hlyE protein, E coli