B-149. The Role of the Undecapeptide in the Pore-Forming Mechanism of the Cholesterol-Dependent Cytolysins

K. J. Dowd, E. M. Hotze, R. K. Tweten;
Univ. of Oklahoma Hlth. Sci. Ctr., Oklahoma City, OK.

The cholesterol-dependent cytolysins (CDCs) are a family of toxins produced by many species of gram positive bacteria. The members of this family exhibit a high degree of sequence homology and structural similarity, with the most highly conserved region in the CDC structures being the undecapeptide sequence at the tip of domain 4 (D4). It has been a long held view that the undecapeptide mediates the interaction of the CDCs with cholesterol-rich membranes, but was recently shown that this function resides in 3 short loops that are juxtaposed to the undecapeptide at the tip of domain 4 (Soltani, 2007. PNAS 104: 20226-20231). Only after the cholesterol-dependent insertion of these loops does the undecapeptide insert into the membrane, leaving the role of the conserved undecapeptide sequence unexplained. Studies herein confirmed these previous findings and provide additional details on the role of specific undecapeptide residues in the cytolytic mechanism of the CDCs. Using a combination of fluorescence spectroscopy, site-specific mutagenesis and gel analysis we have found that mutation of different key residues of the undecapeptide result in cytolytically inactive toxin. Surprisingly, the mutants appear to be affected in different stages of the pore forming mechanism. Mutation of the conserved undecapeptide cysteine (C459) to threonine prevented the membrane insertion of the undecapeptide tryptophans and blocked pore formation, but did not affect assembly of the SDS-resistant prepore oligomer. A similar phenotype is seen when the first of the three conserved tryptophans in the undecapeptide (W464) is mutated to alanine. Interestingly, individual mutation to alanine of the either of the other 2 undecapeptide tryptophan residues (W466 and W467) appears to affect formation of the SDS-resistant prepore oligomer on membranes as there is less (W466A) or no (W467A) detectable oligomer when analyzed by SDS-AGE (sodium dodecyl-sulfate agarose gel electrophoresis). These studies provide important insights into how the undecapeptide structure influences various stages of the assembly of the pore CDC pore complex.