Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence

We have investigated the substrate specificity of the Escherichia coli hemo lysin transporter system. Translocation of hemolysin is dependent on a C-te rminal signal sequence located within the last 60 amino acids of this prote in. Previous comparative studies of the signal sequence have revealed a con served helix(alpha 1)-linker-helix(alpha 2) motif, suggesting that secondar y structure is important for transport. In this study, we generated three r andom libraries in the alpha 1, linker, and alpha 2 regions, as well as an alpha 1-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 correla tion between the genotype and phenotype of the mutant populations allowed u s to objectively identify any functional features involved. It was found th at the alpha 1-amphiphilic helix and the linker are both important for func tion. To our surprise, the second helix of the conserved structural motif w as not essential for transport. The finding that a predicted amphiphilic he lix and hydrophobicity, rather than primary sequence, contribute to transpo rt in the alpha 1 region allows us to speculate on the mechanism of multipl e substrate recognition. This may have implications for understanding the b road substrate specificity common among other ATP-binding cassette transpor ters.