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The role of the [alpha] 1-helical domain of the signal sequence in hemolysin recognition and transport

University of British Columbia

Hemolysin A (HlyA) is an RTX toxin (Repeats in Toxin) secreted by a transport complex consisting of HlyB, HlyD and TolC proteins. It is a unique transport system, in that the transport of HlyA does not require a periplasmic intermediate and its secretion is directed by an uncleaved C-terminal signal sequence. It is known that the signal sequence of leukotoxin A (LktA, a related toxin from P. hemolytica) can functionally replace the signal sequence of HlyA, even though they share little primary sequence homology. CD and NMR analysis of the C-terminal signal peptides of HlyA and LktA have demonstrated that both form similar secondary structures (helix-strand-helix) in a membrane mimetic environment (Yin et al, 1995; Zhang et ah, 1995). Thus it was postulated that a higher order structure distinguished by the amphiphiphilic helices in the signal sequence is important for recognition and transport of HlyA rather then the primary sequence of the signal sequence (Zhang et al., 1993b). In order to explore this hypothesis in greater detail, I have generated and utilized two libraries, a combinatorial library of sequences predicted to form amphiphilic a-helices and a random library of sequences, to further analyze the role of the structural domains in the HlyA signal sequence. The first library replaced the α₁-helix region of the HlyA signal with a library of presumptive amphiphilic a-helices, and thus maintained the secondary structure while varying the identity of individual amino acids. The isolation of twenty-four α₁-helical variants, based upon their ability to support transport, demonstrated that a conserved primary sequence was not essential for the recognition and transport of HlyA. Mutations of several amino acids, previously thought to be critical to recognition, were also shown by this approach to support HlyA transport. The second library replaced the α₁-helix region of the HlyA signal with a library of random sequences, and thus allowed a variety of secondary structures to form, in order to determine if the secondary structure of the α₁-helix region is required to support transport. In this case, if the isolated variants that are able to support transport form an amphiphilic α-helical structure, it supports the hypothesis that the secondary structure of the a i-region is required for transport. The isolation of 65 variants yielded 4 variants that were able to support transport, 6 variants that were unable to support transport, as well as 55 variants with deletions, mutations, insertions or stop codons that did not conform to the design parameters. The 4 variants that were able to support transport had the expected periodicity of polar and nonpolar residues predicted by Kamtekar et al. to maintain the amphiphilic helical structure of the α₁-helix (1991). These results fully support a model in which Hly A recognition by the transport complex occurs via interactions enabled by the secondary structure of the HlyA C-terminal signal sequence.

Thesis/Dissertation

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2009-05-19

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http://hdl.handle.net/2429/7963

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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