Abstract
Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. They are expressed as soluble monomers that assemble spontaneously into multimeric pores. However, owing to their complexity, the assembly processes have not been resolved in detail for any pore-forming toxin. To determine the assembly mechanism for the ring-shaped, homododecameric pore of the bacterial cytolytic toxin ClyA, we collected a diverse set of kinetic data using single-molecule spectroscopy and complementary techniques on timescales from milliseconds to hours, and from picomolar to micromolar ClyA concentrations. The entire range of experimental results can be explained quantitatively by a surprisingly simple mechanism. First, addition of the detergent n-dodecyl-β-D-maltopyranoside to the soluble monomers triggers the formation of assembly-competent toxin subunits, accompanied by the transient formation of a molten-globule-like intermediate. Then, all sterically compatible oligomers contribute to assembly, which greatly enhances the efficiency of pore formation compared with simple monomer addition.
Highlights
Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells
We overcome this challenge by employing a combination of single-molecule fluorescence spectroscopy and complementary biophysical techniques to investigate the mechanism of pore formation of Cytolysin A (ClyA), a cytolytic toxin expressed in virulent Escherichia coli and Salmonella enterica strains[14,15,16]
In combination with two-focus fluorescence correlation spectroscopy (2f-FCS)[21], stopped-flow circular dichroism (CD) and photo-induced cross-linking[22], our results enabled us to identify the kinetic mechanism for the initial conformational rearrangement in the monomeric protein and the following assembly of the cytolytic ClyA pore complex in the presence of detergent
Summary
Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. The intriguing conformational changes that mediate pore assembly have been of great interest[7,8,9,10,11,12,13], but the mechanisms of the underlying structural rearrangements and steps of pore assembly have remained challenging to elucidate, because the potentially very large number of kinetic intermediates are difficult to resolve experimentally We overcome this challenge by employing a combination of single-molecule fluorescence spectroscopy and complementary biophysical techniques to investigate the mechanism of pore formation of Cytolysin A (ClyA), a cytolytic toxin expressed in virulent Escherichia coli and Salmonella enterica strains[14,15,16]. In combination with two-focus fluorescence correlation spectroscopy (2f-FCS)[21], stopped-flow circular dichroism (CD) and photo-induced cross-linking[22], our results enabled us to identify the kinetic mechanism for the initial conformational rearrangement in the monomeric protein and the following assembly of the cytolytic ClyA pore complex in the presence of detergent
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