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Characterization of Variants of the Pore-Forming Toxin ClyA from Escherichia coli Controlled by a Redox Switch
journal contribution
posted on 2014-10-14, 00:00 authored by Daniel Roderer, Stephan Benke, Marcus Müller, Helene Fäh-Rechsteiner, Nenad Ban, Benjamin Schuler, Rudi GlockshuberThe α-pore-forming toxin Cytolysin
A (ClyA) is responsible
for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is a soluble,
34 kDa monomer that assembles into a dodecameric pore complex in the
presence of membranes or detergent. The comparison of the X-ray structures
of monomeric ClyA and the ClyA protomer in the pore complex revealed
one of the largest conformational transitions observed so far in proteins,
involving the structural rearrangement of more than half of all residues,
which is consistent with the finding that conversion from the monomer
to the assembly competent protomer is rate-limiting for pore assembly.
Here, we introduced artificial disulfide bonds at two distinct sites
into the ClyA monomer that both prevent a specific structural rearrangement
required for protomer formation. Using electron microscopy and hemolytic
activity assays, we show that the engineered disulfides indeed trap
these ClyA variants in an assembly incompetent state. Assembly of
the variants into functional pore complexes can be completely recovered
by disulfide reduction. The assembly kinetics of the ClyA variants
recorded with circular dichroism and fluorescence spectroscopy revealed
the same mechanism of protomer formation that was observed for wild-type
ClyA, proceeding via an intermediate with decreased secondary structure
content.