Abstract
Secretion of the 107-kDa hemolysin A (HlyA) from Escherichia coli is mediated by the membrane proteins hemolysin B and hemolysin D. Hemolysin B is a member of the so-called ATP binding cassette transporter superfamily, which includes the multidrug resistance P-glycoprotein, the cystic fibrosis CFTR protein, and the major histocompatibility complex-associated transporter of antigenic peptides. Recognition of HlyA by the hemolysin B/D transporter is dependent on a signal sequence mapped to the C-terminal 50 or so amino acids of the HlyA molecule. We show that the C-terminal 70 amino acids of leukotoxin from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70-amino acid sequence contains no primary sequence similarity to the HlyA signal sequence; however, structural motifs of helix-turn-helix followed by strand-loop-strand can be deduced for both sequences. We also demonstrate by site-directed mutagenesis that changes to these predicted motifs affect transport function. It thus appears that the transport signal of HlyA may be defined by a higher-order structure and that the hemolysin transporter may recognize a much wider diversity of primary sequences than previously anticipated. This finding may have implications for understanding the basis of substrate specificity of other ATP binding cassette transporters.