Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence.
We have investigated the substrate specificity of the Escherichia coli hemolysin transporter system. Translocation of hemolysin is dependent on a C-terminal signal sequence located within the last 60 amino acids of this protein. Previous comparative studies of the signal sequence have revealed a conserved helix(alpha1)-linker-helix(alpha2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the alpha1, linker, and alpha2 regions, as well as an alpha1-amphiphilic helical library to identify features buried within the structural motif that contribute to transport. Combinatorial variants were generated by altering the primary sequence of specific regions, and correlation between the genotype and phenotype of the mutant populations allowed us to objectively identify any functional features involved. It was found that the alpha1-amphiphilic helix and the linker are both important for function. To our surprise, the second helix of the conserved structural motif was not essential for transport. The finding that a predicted amphiphilic helix and hydrophobicity, rather than primary sequence, contribute to transport in the alpha1 region allows us to speculate on the mechanism of multiple substrate recognition. This may have implications for understanding the broad substrate specificity common among other ATP-binding cassette transporters.
Hui
D
D
British Columbia Cancer Research Centre, Vancouver, British Columbia, V5Z 1L3, Canada.
Morden
C
C
Zhang
F
F
Ling
V
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry*
D006460:Hemolysin Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals