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Functional analysis of the escherichia coli hemolysin signal sequence by random oligonucleotide mutagenesis

University of British Columbia

The E. coli hemolysin is an RTX toxin that is secreted by an ATP-binding cassette transporter complex consisting of HlyB, HlyD, and TolC. Translocation of this protein is dependent on a C-terminal signal sequence located within the last 60 amino acids. Previous studies demonstrated that the signal sequence of a similar toxin, P. haemolytica leukotoxin, could replace that of hemolysin and was efficiently transported. While the two signal sequences share little primary sequence similarities, circular dichroism and ¹⁵N nuclear magnetic resonance revealed a conserved helix-strand-helix motif, leading to the hypothesis that this motif is important for efficient secretion. A previous study using helical variants generated by a combinatorial approach suggested that any one of a large number of sequences that yielded a predicted amphiphilic helix in the upstream helical region is sufficient for transport (Morden, 1998). To further elucidate structural requirements of the signal sequence, the helix-strandhelix motif was divided into three regions (α1-linker- α2) and each was subjected to random oligonucleotide mutagenesis analysis. The last 16 residues of the extreme Cterminus was also analyzed using two contiguous random libraries (Cterml and Cterm2). This approach replaced the target region with a random sequence, and the presence or absence of any critical element(s) within each region was deduced by measuring the level of secretion of the mutant population. Based on the results from the five random libraries and the al helical library, a two-domain functional model of the hemolysin signal sequence was proposed. The first domain consists of the α1 and the linker regions. An amphiphilic helical structure in the al region appears to be both sufficient and required for transport. The second domain involves the last 8 residues of the signal sequence, and hydrophobicity in this region appears to be a major determinant of efficient transport. Connecting the two is a stretch of 19 residues, with no specific requirements. This is a surprise since this stretch of amino acids contains the second helix of the helix-strand-helix motif. Nevertheless, this study supports the model that the important features of the signal sequences are secondary structure (i.e. amphiphilic helix) and general biophysical property (i.e. hydrophobicity) rather than primary sequence.

Thesis/Dissertation

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2009-06-16

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http://hdl.handle.net/2429/9237

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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