TY - JOUR
T1 - Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence
AU - Hui, David
AU - Morden, Carla
AU - Zhang, Fang
AU - Ling, Victor
PY - 2000/1/28
Y1 - 2000/1/28
N2 - 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(α1)-linker-helix(α2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the α1, linker, and α2 regions, as well as an α1- 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 α1-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 α1 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.
AB - 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(α1)-linker-helix(α2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the α1, linker, and α2 regions, as well as an α1- 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 α1-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 α1 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.
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U2 - 10.1074/jbc.275.4.2713
DO - 10.1074/jbc.275.4.2713
M3 - Article
C2 - 10644734
AN - SCOPUS:0034723207
SN - 0021-9258
VL - 275
SP - 2713
EP - 2720
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -