Mutations affecting pore formation by haemolysin from Escherichia coli.
By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9-37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI-DIII) located between amino acids 238-410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498-830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670-830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.
Ludwig
A
A
Institut für Genetik und Mikrobiologie, Universität Würzburg, Federal Republic of Germany.
Schmid
A
A
Benz
R
R
Goebel
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D002872:Chromosome Deletion; D004269:DNA, Bacterial; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers